AgsE-deficient strain

ABSTRACT

The present invention relates to a mutant microbial host cell which is deficient in the production of the AgsE protein or in the production of an homologous thereof if compared with a parent microbial host cell which has not been modified and measured under the same conditions. It has been surprisingly found that when the mutant microbial host cell according to the invention is used in a method to produce a compound of interest, for example an enzyme, an improved yield of said compound is obtained if compared to a method in which a parent host cell which has not been modified is used when measured under the same conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of U.S. patent applicationSer. No. 14/408,043, filed on 15 Dec. 2014 which is a National Stageentry of International Application No. PCT/EP2013/065348, filed 19 Jul.2013, which claims priority to European Patent Application No.12177172.9, filed 19 Jul. 2012 and U.S. Provisional Application No.61/673,596, filed 19 Jul. 2012. The disclosures of the priorityapplications are incorporated in their entirety herein by reference.

BACKGROUND

Field of the Invention

The present invention relates to a mutant microbial host cell which hasbeen modified, preferably in its genome, to result in a deficiency inthe production of a polypeptide, to a method to produce the mutantmicrobial host cell and to a method to produce a compound of interestusing said mutant microbial host cell.

Description of Related Art

Different host cell types may be used for different industrial purposes.For example: mammalian cell lines are used for antibody production;fungal cells are preferred organisms for production of polypeptides andsecondary metabolites; bacterial cells are preferred for smallmetabolite and antibiotic production; and plant cells are preferred fortaste and flavor compounds.

Recombinant techniques are widely employed for optimization of theproductivity of such host cells and/or the processes in which they areused. This can involve a multitude of options.

Some techniques will aim at the over expression of a gene of interestcoding for a compound of interest in the host cell. Gene expression canbe modulated in several ways.

For example the gene of interest can be placed in the host cell underthe expression control of a strong promoter, suitable for said cell. Thelatter can occur by introducing an expression cassette into the hostcell, by plasmid- or vector-mediated transformation. Production of thecompound of interest may then be achieved by culturing the transformedhost cell under inducing conditions necessary for the proper functioningof the promoter contained in the expression cassette. For example U.S.Pat. No. 5,722,8547 describes the use of DNA constructs used fortransforming an Aspergillus to obtain expression therein of apolypeptide in which the DNA construct comprises an inducible promoterDNA for promoting transcription in Aspergillus and operably linked to aDNA coding for said polypeptide.

It is known that transcriptional activators are regulatory proteinsfacilitating the binding of RNA polymerase to a promoter controllingexpression of a gene of interest. Gene expression can be modulated byfor example using mutant host cells which produce a specifictranscriptional activator in higher quantities, leading to increasedexpression of a gene of interest which is under the control of apromoter activated by said transcriptional activator. Such an approachis e.g. described in WO2006/040312, referring to the PrtTtranscriptional activator and its use.

In yet an alternative approach gene expression can be improved byincreasing the copy number of the gene of interest in the host cell usedto express the gene. However the number of gene copies present in thehost cell is a limiting factor as recombinant host cells comprising ahigh number of copies of a gene to be expressed may become unstable. Asolution to this problem is given in WO9846772 which describes stablefilamentous fungi comprising multiple substantially homologous DNAdomains wherein in at least 2 of said domains an integrated copy of arecombinant DNA molecule coding for a compound of interest is present.

Yet other approaches aiming at improving the productivity of a compoundof interest by a host cell can involve deletion or inactivation ofcompeting pathways, changing compartmentalization of enzymes, increasingprotein or metabolite secretion, increasing organelle content and thelike.

Despite of advances in the understanding of expression of compounds ofinterests in host cells, there remains a need for methods to increaseproduction of important compounds of interest on commercial orindustrial scale. Surprisingly we have found that the down-regulation ofthe agsE gene in a microbial host cell, for example a filamentous fungalhost cell expressing a compound of interest, e.g. en enzyme of interest,resulted in an increased production of said enzyme by said host cell.

SUMMARY

The present invention relates to a mutant microbial host cell which hasbeen modified, preferably in its genome, to result in a deficiency inthe production of a polypeptide selected from the group consisting of:

-   -   a. a polypeptide according to SEQ ID NO: 3 or a polypeptide at        least 70% identical thereto, preferably a polypeptide at least        70% identical thereto having at least one activity of the        polypeptide according to SEQ ID NO:3;    -   b. a mature polypeptide comprised in SEQ ID NO: 3 or a        polypeptide at least 70% identical thereto, preferably a        polypeptide at least 70% identical thereto having at least one        activity of the mature polypeptide comprised in SEQ ID NO:3;    -   c. a polypeptide encoded by a polynucleotide according to SEQ ID        NO: 1 or 2 or encoded by a polynucleotide at least 70% identical        to SEQ ID NO: 1 or 2, wherein said polypeptide encoded by a        polynucleotide according to SEQ ID NO: 1 or 2 has preferably at        least one activity of the polypeptide encoded by SEQ ID NO: 1 or        2;    -   d. a polypeptide encoded by a polynucleotide capable of        hybridising to a polynucleotide according to SEQ ID NO: 1 or 2        or capable of hybridising to the complementary strand of SEQ ID        NO: 1 or 2, wherein said polypeptide has preferably at least one        activity of the polypeptide encoded by SEQ ID NO: 1 or 2;        if compared with a parent microbial host cell which has not been        modified and measured under the same conditions.

The present invention further relates to a method of producing a mutantmicrobial host cell according to the invention comprising the steps of:

-   -   a. providing a parent microbial host cell;    -   b. modifying the parent microbial host cell, preferably        modifying the genome of the parent microbial host cell to yield        a mutant microbial host cell which is deficient in the        production of a polypeptide selected from the group consisting        of:        -   (i) a polypeptide according to SEQ ID NO: 3 or a polypeptide            at least 70% identical thereto, preferably a polypeptide at            least 70% identical thereto having at least one activity of            the polypeptide according to SEQ ID NO:3;        -   (ii) a mature polypeptide comprised in SEQ ID NO: 3 or a            polypeptide at least 70% identical thereto, preferably a            polypeptide at least 70% identical thereto having at least            one activity of the mature polypeptide comprised in SEQ ID            NO:3;        -   (iii) a polypeptide encoded by a polynucleotide according to            SEQ ID NO: 1 or 2 or encoded by a polynucleotide at least            70% identical to SEQ ID NO: 1 or 2, wherein said polypeptide            encoded by a polynucleotide according to SEQ ID NO: 1 or 2            has preferably at least one activity of the polypeptide            encoded by SEQ ID NO: 1 or 2;        -   (iv) a polypeptide encoded by a polynucleotide capable of            hybridising to a polynucleotide according to SEQ ID NO: 1 or            2 or capable of hybridising to the complementary strand of            SEQ ID NO: 1 or 2, wherein said polypeptide has preferably            at least one activity of the polypeptide encoded by SEQ ID            NO: 1 or 2;            if compared with the parent microbial host cell and measured            under the same conditions.

The invention relates as well to a method for the production of acompound of interest by microbial fermentation comprising:

-   -   a. providing a mutant microbial host cell according to the        invention or produced according to a method for producing a        mutant microbial host cell according to the invention capable of        expressing the compound of interest,    -   b. culturing said microbial host cell under conditions conducive        to the expression of the compound of interest,    -   c. optionally isolating the compound of interest from the        culture medium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts pGBTOPGOX-3, the pGBTOP-12 based plasmid used forexpression of the Penicillium chrysogenum glucose oxidase enzyme genewith a layout for expression driven by the glucoamylase promoter andtargeted integration in the adapted BamHI amplicon.

FIG. 2 depicts pGBTOPLIP-2, the pGBTOP-12 based plasmid used forexpression of the L01 lipase enzyme (as described in WO2009/106575),with the L01 gene cloned in it and with a layout for expression drivenby the glucoamylase promoter and targeted integration in the adaptedBamHI amplicon.

FIG. 3 depicts pGBTOPPLA-2, the pGBTOP-12 based plasmid used forexpression of the porcine phospholipase A2 (PLA2) enzyme, with GLA-PLA2encoding gene cloned in it and with a layout for expression driven bythe glucoamylase promoter and targeted integration in the adapted BamHIamplicon.

FIG. 4 depicts pGBDEL-AMY1, the plasmid used for deletion of the amylaseencoding agdB gene with a layout representative for other deletionconstructs (i.e. pGBDEL-AMY2, and pGBDEL-AMY4)

FIG. 5 depicts relative glucose oxidase activities, as measured in theculture supernatant of the different strains. The activity of the PGOX-2reference strain at day 4 was set at a level of 100%.

FIG. 6 depicts glucose oxidase activities on plate of the differentmutant strains. Growth was on 1% maltose and staining with o-anisidinewas done after 4 days of growth.

FIG. 7 depicts relative lipase activities, as measured in the culturesupernatant at day 4 of the different strains as indicated. The activityof one of the two LIP2 reference strains was set at a level of 100%.

FIG. 8 depicts relative PLA2 activities, as measured in the culturesupernatant after 5 days of fermentation of the strains as indicated.The activity of the PLA2 reference strain was set at a level of 100%.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the genomic sequence of the agsE gene fromAspergillus niger, including 2 kb upstream and downstream flankingregions. The genomic sequence comprises the cDNA sequence according toSEQ ID NO: 2.

SEQ ID NO: 2 sets out the cDNA sequence of the agsE gene from A. niger.

SEQ ID NO: 3 sets out the amino acid sequence of the AgsE protein fromA. niger.

SEQ ID NO: 4 sets out the amino acid sequence of the mature AgsE proteincorresponding to amino acid 20-2426 of SEQ ID NO: 3.

SEQ ID NO: 5 sets out the genomic sequence of the agdB gene fromAspergillus niger, including 2 kb upstream and downstream flankingregions. The genomic sequence comprises the cDNA sequence according toSEQ ID NO: 6.

SEQ ID NO: 6 sets out the cDNA sequence of the agdB gene from A. niger.

SEQ ID NO: 7 sets out the amino acid sequence of the AgdB protein fromA. niger.

SEQ ID NO: 8 sets out the genomic sequence of the agdA gene fromAspergillus niger, including 2 kb upstream and downstream flankingregions. The genomic sequence comprises the cDNA sequence according toSEQ ID NO: 9.

SEQ ID NO: 9 sets out the cDNA sequence of the agdA gene from A. niger.

SEQ ID NO: 10 sets out the amino acid sequence of the AgdA protein fromA. niger.

SEQ ID NO: 11 sets out the codon pair optimized cDNA sequence of theglucose oxidase from Penicillium chrysogenum

SEQ ID NO: 12 sets out the amino acid sequence of the glucose oxidasefrom Penicillium chrysogenum.

SEQ ID NO: 13 sets out the genomic sequence of the amyC amylase genefrom Aspergillus niger, including 2 kb upstream and downstream flankingregions. The genomic sequence comprises the cDNA sequence according toSEQ ID NO: 2.

SEQ ID NO: 14 sets out the cDNA sequence of the amyC amylase gene (shortsequence) from A. niger.

SEQ ID NO: 15 sets out the amino acid sequence of the amyC amylaseprotein (short sequence) from A. niger.

SEQ ID NO: 16 sets out the amino acid sequence of the AmyC matureamylase protein (short sequence) corresponding to amino acid 17-493 ofSEQ ID NO: 15.

SEQ ID NO: 17 sets out the cDNA sequence of the amyC amylase gene (longsequence) from A. niger.

SEQ ID NO: 18 sets out the amino acid sequence of the amyC amylaseprotein (long sequence) from A. niger.

SEQ ID NO: 19 sets out the amino acid sequence of the AmyC matureamylase protein (long sequence) corresponding to amino acid 17-524 ofSEQ ID NO: 18.

SEQ ID NO: 20 sets out the amino acid sequence of a fusion proteincomprising a native glucoamylase A gene of A. niger fused with proPLA2(porcine phospholipase A2) fused.

All nucleotide sequences for A. niger genes and protein sequences andtheir genomic context can be derived from public databases available forexample from the NCBI at ncbi.nlm.nih.gov/ or EMBL (ebi.ac.uk/embl/).For example the genome sequence of CBS 513.88 at EMBL has accessionnumbers no. AM269948-AM270415.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

The present invention relates to a mutant microbial host cell which hasbeen modified, preferably in its genome, to result in a deficiency inthe production of a polypeptide selected from the group consisting of:

-   -   a. a polypeptide according to SEQ ID NO: 3 or a polypeptide at        least 70% identical thereto, and preferably having at least one        activity of the polypeptide according to SEQ ID NO:3;    -   b. a mature polypeptide comprised in SEQ ID NO: 3 or a        polypeptide at least 70% identical thereto, and preferably        having at least one activity of the mature polypeptide comprised        in SEQ ID NO:3;    -   c. a polypeptide encoded by a polynucleotide according to SEQ ID        NO: 1 or 2 or encoded by a polynucleotide at least 70% identical        to SEQ ID NO: 1 or 2, wherein said polypeptide encoded by a        polynucleotide according to SEQ ID NO: 1 or 2 has preferably at        least one activity of the polypeptide encoded by SEQ ID NO: 1 or        2;    -   d. a polypeptide encoded by a polynucleotide capable of        hybridising to a polynucleotide according to SEQ ID NO: 1 or 2        or capable of hybridising to the complementary strand of SEQ ID        NO: 1 or 2, wherein said polypeptide has preferably at least one        activity of the polypeptide encoded by SEQ ID NO: 1 or 2;    -   if compared with a parent microbial host cell which has not been        modified and measured under the same conditions.

It has been surprisingly found that when the mutant microbial host cellaccording to the invention and which is capable of expressing a compoundof interest is used in a method to produce a compound of interest, forexample an enzyme, an improved yield of said compound is obtained ifcompared to a method in which a parent host cell is used and measuredunder the same conditions.

In addition, it has been found that when the mutant microbial host cellaccording to the invention and which is capable of expressing a compoundof interest is used in a method to produce a compound of interest, thefermentation broth comprising the mutant microbial host celldemonstrates a remarkably low viscosity. This property is especiallyrelevant for an industrial scale process since it allows a fermentationprocess in which a mutant microbial host cell of the invention is usedto be carried out using less energy or carried out more intensively.

Within the context of the present invention “measured under the sameconditions” or “analysed under the same conditions” means that themutated microbial host cell and the parent microbial host cell arecultivated under the same conditions and that the amount and/or activityof the polypeptide in which the mutant host cell is deficient, ifcompared to the parent microbial host cell, is measured in the microbialhost cell and in the parent host cell, respectively, using the sameconditions, preferably by using the same assay and/or methodology, morepreferably within the same experiment.

A “mutant microbial host cell” is herewith defined as a microbial hostcell derived from a parent host cell and which has been modified,preferably in its genome, if compared to the parent host cell to obtaina different genotype and/or a different phenotype if compared to theparent host cell from which it is derived.

The modification can either be effected by

-   -   a) subjecting the parent microbial host cell to recombinant        genetic manipulation techniques; and/or    -   b) subjecting the parent microbial host cell to (classical)        mutagenesis; and/or    -   c) subjecting the parent microbial host cell to an inhibiting        compound or composition.

A “mutant microbial host cell which has been modified, preferably in itsgenome, to result in a deficiency in the production of a product”, forexample of a product such as a polypeptide according to SEQ ID NO: 3, isherein defined as a mutant microbial host cell which has been modified,preferably in its genome, to result in a phenotypic feature wherein thecell: a) produces less of the product or produces substantially noproduct and/or b) produces a product having a decreased activity ordecreased specific activity or a product having no activity or nospecific activity and combinations of one or more of these possibilitiesas compared to the parent microbial host cell that has not beenmodified, when analysed under the same conditions.

In the context of the present invention the mutant microbial host cellaccording to the invention is deficient in the production of apolypeptide. Said polypeptide has preferably an enzymatic activity whichis preferably a glycoside hydrolase activity, more preferably anenzymatic activity selected from the group consisting of: α-amylaseactivity [EC 3.2.1.1], isoamylase activity, inulinase activity,invertase activity [EC 3.2.1.26], maltase activity [EC 3.2.1.20],isomaltase activity, pullulanase activity, glucoamylase activity,cyclodextrinase activity, chitosanase activity, dextranase activity,sucrase-isomaltase activity, α-glucosidase activity, glycogendebranching enzymatic activity.

In another embodiment said polypeptide has preferably an enzymaticactivity which is α-gluconotransferase activity, an enzymatic activitywhich is preferably a glycoside transferase or glycoside synthaseactivity, more preferably an enzymatic activity selected from the groupconsisting of: glycogen branching enzymatic activity, α-1,3-glucansynthase enzymatic activity [EC 2.4.1.183], α-1,4-glucan synthaseactivity, α-1,6-glucan synthase activity, β-1,3-glucan synthaseactivity, β-1,4-glucan synthase activity, β-1,6-glucan synthaseactivity, glucoamylase activity, maltopentaose-forming amylase activity,maltohexaose-forming amylase activity, α-glucosidase activity,α-glucosidase II activity, α-xylosidase activity.

This polypeptide is selected from the group consisting of:

-   -   a. a polypeptide according to SEQ ID NO: 3 or a polypeptide at        least 70% identical thereto and preferably having at least one        activity of the polypeptide according to SEQ ID NO:3;    -   b. a mature polypeptide comprised in SEQ ID NO: 3 or a        polypeptide at least 70% identical thereto preferably having at        least one activity of the mature polypeptide comprised in SEQ ID        NO:3;    -   c. a polypeptide encoded by a polynucleotide according to SEQ ID        NO: 1 or 2 or encoded by a polynucleotide at least 70% identical        to SEQ ID NO: 1 or 2, wherein said polypeptide encoded by a        polynucleotide according to SEQ ID NO: 1 or 1 has preferably at        least one activity of the polypeptide encoded by SEQ ID NO: 1 or        2;    -   d. a polypeptide encoded by a polynucleotide capable of        hybridising to a polynucleotide according to SEQ ID NO: 1 or 2        or capable of hybridising to the complementary strand of SEQ ID        NO: 1 or 2, wherein said polypeptide has preferably at least one        activity of the polypeptide encoded by SEQ ID NO: 1 or 2;

A polypeptide according to SEQ ID NO: 3 corresponds to the putativeα-1,3-D-glucan synthase agsE from Aspergillus niger (Yuan X.-L., van derKaaij R. M., van den Hondel C. A. M. J. J., Punt P. J., van der Marel M.J. E. C., Dijkhuizen L., Ram A. F. J. Mol. Genet. Genomics (2008) 279:545-561). The polypeptide according to SEQ ID NO: 3 is encoded by theagsE gene (genomic DNA as depicted in SEQ ID NO:1, cDNA as depicted inSEQ ID NO: 2).

In the context of the present invention a polypeptide which is at least70% identical to SEQ ID NO: 3 is a polypeptide characterized by an aminoacid sequence comprising one or more substitutions, deletions, and/orinsertions of one or more amino acids if compared to the polypeptide ofSEQ ID NO: 3 and which has preferably at least one enzymatic activity ofthe polypeptide according to SEQ ID NO: 3. Therefore the polypeptideaccording to SEQ ID NO: 3 and a polypeptide at least 70% identicalthereto have preferably at least one enzymatic activity in common. Saidat least one enzymatic activity is preferably a glycoside hydrolaseactivity, more preferably an enzymatic activity selected from the groupconsisting of: α-amylase activity [EC 3.2.1.1], isoamylase activity,inulinase activity, invertase activity [EC 3.2.1.26], maltase activity[EC 3.2.1.20], isomaltase activity, pullulanase activity, glucoamylaseactivity, cyclodextrinase activity, chitosanase activity, dextranaseactivity, sucrase-isomaltase activity, α-glucosidase activity, glycogendebranching enzymatic activity.

In another embodiment said enzymatic activity is: α-gluconotransferaseactivity, enzymatic activity is preferably a glycoside transferase orglycoside synthase activity, more preferably an enzymatic activityselected from the group consisting of: glycogen branching enzymaticactivity, α-1,3-glucan synthase enzymatic activity [EC 2.4.1.183],α-1,4-glucan synthase activity, α-1,6-glucan synthase activity,β-1,3-glucan synthase activity, β-1,4-glucan synthase activity,β-1,6-glucan synthase activity, glucoamylase activity,maltopentaose-forming amylase activity, maltohexaose-forming amylaseactivity, α-glucosidase activity, α-glucosidase II activity,α-xylosidase activity.

The polypeptide which is at least 70% identical to SEQ ID NO: 3 andhaving at least one (enzymatic) activity of the polypeptide according toSEQ ID NO: 3 may have more or less of said at least one activity thanthe polypeptide according to SEQ ID NO: 3. The polypeptide which is atleast 70% identical to SEQ ID NO: 3 may e.g. be a natural variant, anorthologue or an in vitro generated variant of SEQ ID NO: 3 obtainedusing methods well known in the art such as e.g. classical mutagenesis,site-directed mutagenesis, DNA shuffling and in silico design. In thecontext of the present invention the polypeptide which is at least 70%identical to SEQ ID NO: 3 has preferably between 20% and 400% enzymaticactivity if compared to SEQ ID NO:3 and measured under the sameconditions, more preferably between 40 and 350% activity, even morepreferably between 50 and 300% activity, between 70 and 250% activity,between 80 and 200% activity, most preferably approximately 100%activity of the polypeptide according to SEQ ID NO: 3. With activity itis herewith intended an enzymatic activity as mentioned above. For themeasurement of the at least one enzymatic activity in the polypeptideaccording to SEQ ID NO: 3 and in the polypeptide at least 70% identicalthereto and having at least one enzymatic activity of the polypeptideaccording to SEQ ID NO: 3 any method known in the art for themeasurement of said specific activity can be used. The only requirementis that the measurement of said activity in the polypeptide according toSEQ ID NO: 3 and in the polypeptide at least 70% identical thereto isperformed using the same method and/or assay and under the sameconditions, preferably within the same experiment. α-amylase activitycan preferably be measured according to the well-established Ceralphamethod for the determination of α-amylase activity described in theexperimental session. α-1,3-glucan synthase activity can be measuredaccording to the method described in “Tsumori H., Shimamura A., MukasaH. Journal of General Microbiology (1985) 131: 553-559”. Preferably thepolypeptide is at least 80% identical to SEQ ID NO: 3, more preferablyat least 85% identical to SEQ ID NO: 3, even more preferably at least90% identical to SEQ ID NO: 3, most preferably at least 91%, for exampleat least 92%, 93%, 94%, at least 95% identical, at least 96%, 97%, 98%,at least 99% identical to SEQ ID NO: 3. Preferably the polypeptide is apolypeptide according to SEQ ID NO: 3. Preferably sequence identity ismeasured over the whole polypeptide sequence length.

The polypeptide which production the mutant microbial host cellaccording to the invention is deficient in, may be a mature polypeptidecomprised in SEQ ID NO: 3. A mature polypeptide is defined herein as apolypeptide in its final form after translation, post-translationalmodifications, such as N-terminal processing, C-terminal processing,glycosylation, phosphorylation, secretion and optional removal of leadersequences by (proteolytic) cleavage. Signal peptides, propeptides andprepropeptides are in the art sometimes referred to as “leadersequences”. The term “propeptide” is defined herein as a peptide fusedin frame to the N-terminus of a polypeptide having biological activity.The resulting polypeptide is known as a propolypeptide which is lackingthe polypeptide biological activity and can be converted into a mature,biologically active, polypeptide by catalytic or autocatalytic cleavageof the propeptide from the propolypeptide. A signal peptide andpropeptide together are herein referred to as a “prepropeptide”. The“signal sequence” is defined herein as a peptide being fused in frame tothe N-terminus of a propeptide and the propeptide being fused in frameto the N-terminus of a polypeptide having biological activity. In somecases the propeptide is lacking and the signal sequence is fused inframe to the N-terminus of the polypeptide. The function of the signalsequence is to direct the polypeptide into the cell secretory pathway.

Therefore SEQ ID NO: 3 may be the sequence translated from the mRNA andprior to post translational modifications. SEQ ID NO: 3 may compriseadditional amino acids at either the C-terminus and/or the N-terminus ifcompared to the mature polypeptide comprised therein. SEQ ID NO: 3 maye.g. comprise the mature polypeptide linked in frame to its signalpeptide, propeptide and/or prepropeptide. In a preferred embodiment themature polypeptide comprised in SEQ ID NO: 3 corresponds to amino acids20-2426 of SEQ ID NO: 3 and is set out in SEQ ID NO: 4. Therefore in oneembodiment the mutant microbial host cell according to the invention isdeficient in a polypeptide which is the mature polypeptide according toSEQ ID NO: 4.

In the context of the present invention the polypeptide which productionthe mutant microbial cell is deficient in may be a polypeptide at least70% identical to the mature polypeptide as defined herein and havingpreferably at least one activity as defined herein of said maturepolypeptide. Therefore the mature polypeptide comprised in SEQ ID NO: 3as defined herein, preferably a mature polypeptide according to SEQ IDNO: 4 and the polypeptide at least 70% identical thereto have preferablyat least one enzymatic activity in common. Said at least one enzymaticactivity is preferably a glycoside hydrolase activity, more preferablyan enzymatic activity selected from the group consisting of: α-amylaseactivity [EC 3.2.1.1], isoamylase activity, inulinase activity,invertase activity [EC 3.2.1.26], maltase activity [EC 3.2.1.20],isomaltase activity, pullulanase activity, glucoamylase activity,cyclodextrinase activity, chitosanase activity, dextranase activity,sucrase-isomaltase activity, α-glucosidase activity, glycogendebranching enzymatic activity.

In another embodiment said enzymatic activity is: α-gluconotransferaseactivity, enzymatic activity is preferably a glycoside transferase orglycoside synthase activity, more preferably an enzymatic activityselected from the group consisting of: glycogen branching enzymaticactivity, α-1,3-glucan synthase enzymatic activity [EC 2.4.1.183],α-1,4-glucan synthase activity, α-1,6-glucan synthase activity,β-1,3-glucan synthase activity, β-1,4-glucan synthase activity,β-1,6-glucan synthase activity, glucoamylase activity,maltopentaose-forming amylase activity, maltohexaose-forming amylaseactivity, α-glucosidase activity, α-glucosidase II activity,α-xylosidase activity.

Preferably the polypeptide is at least 80% identical to the maturepolypeptide as defined herein, more preferably at least 85% identical tothe mature polypeptide as defined herein, even more preferably at least90% identical to the mature polypeptide as defined herein, mostpreferably at least 91%, for example at least 92%, 93%, 94%, at least95% identical, at least 96%, 97%, 98%, at least 99% identical to themature polypeptide as defined herein. Preferably the polypeptide is themature polypeptide according to SEQ ID NO: 4. Preferably sequenceidentity is measured over the whole polypeptide sequence length.

In the context of the present invention a polynucleotide according toSEQ ID NO: 1 or 2 or a polynucleotide at least 70% identical to SEQ IDNO: 1 or 2 is a polynucleotide coding for a polypeptide according to SEQID NO: 3, for a mature polypeptide comprised in SEQ ID NO: 3, for apolypeptide according to SEQ ID NO: 4 or for a polypeptide having atleast 70% identity to SEQ ID NO: 3, for a polypeptide having at least70% identity to a mature polypeptide comprised in SEQ ID NO: 3, for apolypeptide having at least 70% identity to a polypeptide according toSEQ ID NO: 4 as defined above. In the context of the present invention apolynucleotide at least 70% identical to SEQ ID NO: 1 or 2 is apolynucleotide characterized by a nucleotide sequence comprising one ormore substitutions, deletions, and/or insertions of one or morenucleotides if compared to the polynucleotide of SEQ ID NO: 1 or 2.Preferably the polynucleotide is at least 80% identical to SEQ ID NO: 1or 2, more preferably at least 85% identical to SEQ ID NO: 1 or 2, evenmore preferably at least 90% identical to SEQ ID NO: 1 or 2, mostpreferably at least 91%, 92%, 93%, 94%, at least 95% identical, at least96%, 97%, 98%, at least 99% identical to SEQ ID NO: 1 or 2. Preferablythe polynucleotide is a polynucleotide according to SEQ ID NO: 1 or 2.Preferably the polypeptide encoded by a polynucleotide at least 70%identical to SEQ ID NO: 1 or 2 has at least one enzymatic activity asdefined herein of the polypeptide encoded by SEQ ID NO: 1 or 2.Therefore the polypeptide encoded by SEQ ID NO: 1 or 2 and a polypeptideencoded by a polynucleotide at least 70% identical to SEQ ID NO: 1 or 2have at least one enzymatic activity in common. Said at least oneenzymatic activity is preferably a glycoside hydrolase activity, morepreferably an enzymatic activity selected from the group consisting of:α-amylase activity [EC 3.2.1.1], isoamylase activity, inulinaseactivity, invertase activity [EC 3.2.1.26], maltase activity [EC3.2.1.20], isomaltase activity, pullulanase activity, glucoamylaseactivity, cyclodextrinase activity, chitosanase activity, dextranaseactivity, sucrase-isomaltase activity, α-glucosidase activity, glycogendebranching enzymatic activity.

In another embodiment said enzymatic activity is: α-gluconotransferaseactivity, enzymatic activity is preferably a glycoside transferase orglycoside synthase activity, more preferably an enzymatic activityselected from the group consisting of: glycogen branching enzymaticactivity, α-1,3-glucan synthase enzymatic activity [EC 2.4.1.183],α-1,4-glucan synthase activity, α-1,6-glucan synthase activity,β-1,3-glucan synthase activity, β-1,4-glucan synthase activity,β-1,6-glucan synthase activity, glucoamylase activity,maltopentaose-forming amylase activity, maltohexaose-forming amylaseactivity, α-glucosidase activity, α-glucosidase II activity,α-xylosidase activity.

For the purpose of this invention, it is defined here that in order todetermine the percentage of sequence identity of two amino acidsequences or of two nucleic acid sequences, the sequences are alignedfor optimal comparison purposes. In order to optimize the alignmentbetween the two sequences gaps may be introduced in any of the twosequences that are compared. Such alignment can be carried out over thefull length of the sequences being compared. Alternatively, thealignment may be carried out over a shorter length, for example overabout 20, about 50, about 100 or more nucleic acids/based or aminoacids. The sequence identity is the percentage of identical matchesbetween the two sequences over the reported aligned region.

A comparison of sequences and determination of percentage of sequenceidentity between two sequences can be accomplished using a mathematicalalgorithm. The skilled person will be aware of the fact that severaldifferent computer programs are available to align two sequences anddetermine the identity between two sequences (Kruskal, J. B. (1983) Anoverview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.),Time warps, string edits and macromolecules: the theory and practice ofsequence comparison, pp. 1-44 Addison Wesley). The percentage ofsequence identity between two amino acid sequences or between twonucleotide sequences may be determined using the Needleman and Wunschalgorithm for the alignment of two sequences. (Needleman, S. B. andWunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). Both amino acidsequences and nucleotide sequences can be aligned by the algorithm. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this invention the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp 276-277,emboss.bioinformatics.nl/). For protein sequences EBLOSUM62 is used forthe substitution matrix. For nucleotide sequence, EDNAFULL is used. Theoptional parameters used are a gap-open penalty of 10 and a gapextension penalty of 0.5. The skilled person will appreciate that allthese different parameters will yield slightly different results butthat the overall percentage identity of two sequences is notsignificantly altered when using different algorithms.

After alignment by the program NEEDLE as described above the percentageof sequence identity between a query sequence and a sequence of theinvention is calculated as follows: Number of corresponding positions inthe alignment showing an identical amino acid or identical nucleotide inboth sequences divided by the total length of the alignment aftersubtraction of the total number of gaps in the alignment. The identitydefined as herein can be obtained from NEEDLE by using the NOBRIEFoption and is labeled in the output of the program as“longest-identity”.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the homepage of the NationalCenter for Biotechnology Information at ncbi.nlm.nih.gov/.

In the context of the present invention a polypeptide which productionthe mutant microbial host cell according to the invention may bedeficient in, may be a polypeptide encoded by a polynucleotide capableof hybridising to SEQ ID NO: 1 or 2 or capable of hybridising to thecomplementary strand of a polynucleotide according to SEQ ID NO: 1 or 2,preferably it is capable of hybridising under low stringency conditions,more preferably it is capable of hybridising under medium stringencyconditions, even more preferably it is capable of hybridising under highstringency conditions to the complementary strand of a polynucleotideaccording to SEQ ID NO: 1 or 2. Preferably a polypeptide encoded by apolynucleotide capable of hybridising to SEQ ID NO: 1 or 2 or capable ofhybridising to the complementary strand of a polynucleotide according toSEQ ID NO: 1 or 2 has at least one enzymatic activity in common with thepolypeptide encoded by SEQ ID NO: 1 or 2. Said at least one enzymaticactivity is preferably a glycoside hydrolase activity, more preferablyan enzymatic activity selected from the group consisting of: α-amylaseactivity [EC 3.2.1.1], isoamylase activity, inulinase activity,invertase activity [EC 3.2.1.26], maltase activity [EC 3.2.1.20],isomaltase activity, pullulanase activity, glucoamylase activity,cyclodextrinase activity, chitosanase activity, dextranase activity,sucrase-isomaltase activity, α-glucosidase activity, glycogendebranching enzymatic activity.

In another embodiment said enzymatic activity is: α-gluconotransferaseactivity, enzymatic activity is preferably a glycoside transferase orglycoside synthase activity, more preferably an enzymatic activityselected from the group consisting of: glycogen branching enzymaticactivity, α-1,3-glucan synthase enzymatic activity [EC 2.4.1.183],α-1,4-glucan synthase activity, α-1,6-glucan synthase activity,β-1,3-glucan synthase activity, β-1,4-glucan synthase activity,β-1,6-glucan synthase activity, glucoamylase activity,maltopentaose-forming amylase activity, maltohexaose-forming amylaseactivity, α-glucosidase activity, α-glucosidase II activity,α-xylosidase activity.

As used herein, the term “hybridizing” is intended to describeconditions for hybridization and washing under which polynucleotidesequences at least about 60%, 65%, 80%, 85%, 90%, preferably at least93%, more preferably at least 95% and most preferably at least 98%identical to each other typically remain hybridized to the complement ofeach other. As used herein, the term “hybridization” means the pairingof substantially complementary strands of oligomeric compounds. Onemechanism of pairing involves hydrogen bonding, which may beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleotide bases (nucleotides) of the strands ofoligomeric compounds. For example, adenine and thymine are complementarynucleic acids which pair through the formation of hydrogen bonds.Hybridization can occur under varying circumstances. “Stringencyhybridization” or “hybridizes under low stringency, medium stringency,high stringency, or very high stringency conditions” is used herein todescribe conditions for hybridization and washing, more specificallyconditions under which an oligomeric compound will hybridize to itstarget sequence, but to a minimal number of other sequences. So, theoligomeric compound will hybridize to the target sequence to adetectably greater degree than to other sequences. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6:3.6.

The skilled artisan will know which conditions to apply for low, mediumand high stringency hybridization conditions. Additional guidanceregarding such conditions is readily available in the art, for example,in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Press, N.Y.; and Ausubel et al. (eds.), 1995, CurrentProtocols in Molecular Biology, (John Wiley & Sons, N.Y.).

Stringency conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. Generally, stringency conditions are selected to beabout 5° C. lower than the thermal melting point (T_(m)) for theoligomeric compound at a defined ionic strength and pH. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of anoligomeric compound hybridizes to a perfectly matched probe. Stringencyconditions may also be achieved with the addition of destabilizingagents such as formamide.

Examples of specific hybridization conditions are as follows: 1) lowstringency hybridization conditions in 6× sodium chloride/sodium citrate(SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS atleast at 50° C. (the temperature of the washes can be increased to 55°C. for low stringency conditions); 2) medium stringency hybridizationconditions in 6×SSC at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 60° C.; 3) high stringency hybridization conditionsin 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC,0.1% SDS at 65° C.; and 4) very high stringency hybridization conditionsare 0.5M sodium phosphate, 7% SDS at 65° C., followed by one or morewashes at 0.2×SSC, 1% SDS at 65° C.

Within the context of the present invention the mutant microbial hostcell is deficient in the production of a polypeptide as defined hereinwhen the host cell comprises a modification, preferably in its genome,which results in a reduced or no production of the polypeptide asdefined herein if compared to the parent microbial host cell that hasnot been modified, when analysed under the same conditions and/orcomprises a modification which results in a polypeptide derived from thepolypeptide as described herein with decreased or no (enzymatic)activity (which activity has been defined herein), if compared to theparent microbial host cell that has not been modified, when analysedunder the same conditions. Therefore a mutant microbial host cell asdefined herein is deficient in the production of a polypeptide asdescribed herein when

-   -   a) it produces less polypeptide as defined herein or it produces        no polypeptide as defined herein if compared with the parent        microbial host cell which has not been modified and measured        under the same conditions; and/or    -   b) it produces a polypeptide derived from the polypeptide as        defined herein with decreased or no activity if compared to the        parent microbial host cell that has not been modified, when        analysed under the same conditions.

In one embodiment the mutant microbial host cell produces 1% lesspolypeptide as defined herein if compared with the parent microbial hostcell which has not been modified and measured under the same conditions,at least 5% less, at least 10% less, at least 20% less, at least 30%less, at least 40% less, at least 50% less, at least 60% less, at least70% less, at least 80% less, at least 90% less, at least 91% less, atleast 92% less, at least 93% less, at least 94% less at least 95% less,at least 96% less, at least 97% less, at least 98% less, at least 99%less, or at least 99.9% less. Preferably the mutant microbial host cellproduces substantially no polypeptide as described herein if comparedwith the parent microbial host cell which has not been modified andmeasured under the same conditions.

In one embodiment the mutant microbial host cell produces a polypeptidederived from the polypeptide as defined herein with 1% less (enzymatic)activity as defined herein, if compared with the parent microbial hostcell which has not been modified and measured under the same conditions,at least 5% less activity, at least 10% less activity, at least 20% lessactivity, at least 30% less activity, at least 40% less activity, atleast 50% less activity, at least 60% less activity, at least 70% lessactivity, at least 80% less activity, at least 90% less activity, atleast 91% less activity, at least 92% less activity, at least 93% lessactivity, at least 94% less activity, at least 95% less activity, atleast 96% less activity, at least 97% less activity, at least 98% lessactivity, at least 99% less activity, or at least 99.9% less activity.Preferably the mutant microbial host cell produces a polypeptide derivedfrom a polypeptide as described herein with substantially no activity ifcompared with the parent microbial host cell which has not been modifiedand analysed under the same conditions.

Said enzymatic activity is preferably a glycoside hydrolase activity,more preferably an enzymatic activity selected from the group consistingof: α-amylase activity [EC 3.2.1.1], isoamylase activity, inulinaseactivity, invertase activity [EC 3.2.1.26], maltase activity [EC3.2.1.20], isomaltase activity, pullulanase activity, glucoamylaseactivity, cyclodextrinase activity, chitosanase activity, dextranaseactivity, sucrase-isomaltase activity, α-glucosidase activity, glycogendebranching enzymatic activity.

In another embodiment said enzymatic activity is: α-gluconotransferaseactivity, enzymatic activity is preferably a glycoside transferase orglycoside synthase activity, more preferably an enzymatic activityselected from the group consisting of: glycogen branching enzymaticactivity, α-1,3-glucan synthase enzymatic activity [EC 2.4.1.183],α-1,4-glucan synthase activity, α-1,6-glucan synthase activity,β-1,3-glucan synthase activity, β-1,4-glucan synthase activity,β-1,6-glucan synthase activity, glucoamylase activity,maltopentaose-forming amylase activity, maltohexaose-forming amylaseactivity, α-glucosidase activity, α-glucosidase II activity,α-xylosidase activity.

Deficiency of a mutant microbial host cell according to the invention inthe production of a polypeptide as defined herein may be measured bydetermining the amount and/or (specific) activity of polypeptide havingan enzymatic activity as defined herein produced by the microbial hostcell modified in its genome and/or it may be measured by determining theamount of mRNA transcribed from a polynucleotide encoding thepolypeptide as described herein and/or it may be measured by gene orgenome sequencing if compared to the parent host cell which has not beenmodified.

A modification in the genome can be determined by comparing the DNAsequence of the mutant microbial host cell to the sequence of the parent(non-modified) microbial host cell. Sequencing of DNA and genomesequencing can be done using standard methods known to the personskilled in the art, for example using Sanger sequencing technologyand/or next generation sequencing technologies such as Illumina GA2,Roche 454, etc. as reviewed in Elaine R. Mardis (2008), Next-GenerationDNA Sequencing Methods, Annual Review of Genomics and Human Genetics, 9:387-402. (doi:10.1146/annurev.genom.9.081307.164359)

Deficiency in the production of the polypeptide as described herein canbe measured using any assay suitable to the measurement of thepolypeptide enzymatic activity as defined herein available to theskilled person, transcriptional profiling, Northern blotting RT-PCR,Q-PCR and Western blotting. In particular quantifying the amount of mRNApresent in a cell may for example be achieved by northern blotting (inMolecular Cloning: A Laboratory Manual, Sambrook et al., New York: ColdSpring Harbour Press, 1989). Quantifying the amount of polypeptide asdescribed herein present in a cell may for example be achieved bywestern blotting. The difference in mRNA amount may also be quantifiedby DNA array analysis (Eisen, M. B. and Brown, P. O. DNA arrays foranalysis of gene expression. Methods Enzymol. 1999, 303:179-205).

A modification, preferably in the genome, is construed as one or moremodifications.

The modification, preferably in the genome, can either be effected by

-   -   a) subjecting the parent microbial host cell to recombinant        genetic manipulation techniques; and/or    -   b) subjecting the parent microbial host cell to (classical)        mutagenesis; and/or    -   c) subjecting the parent microbial host cell to an inhibiting        compound or composition.

Modification of a genome of a (mutant) microbial host cell is hereindefined as any event resulting in a change in a polynucleotide sequencein the genome of the cell. In a preferred embodiment the mutantmicrobial host cell according to the invention has a modification,preferably in its genome comprising:

-   -   a) a modification which results in a reduced or no production of        a polypeptide as defined herein if compared to the parent        microbial host cell that has not been modified, when analysed        under the same conditions and/or    -   b) a modification which results in a polypeptide derived from a        polypeptide as defined herein with decreased or no (enzymatic)        activity as defined herein if compared to the parent microbial        host cell that has not been modified, when analysed under the        same conditions.

Modification can be introduced by classical strain improvement, randommutagenesis followed by selection. Modification can also be introducedby site-directed mutagenesis.

Modification may be accomplished by the introduction (insertion),substitution (replacement) or removal (deletion) of one or morenucleotides in a polynucleotide sequence. A full or partial deletion ofa polynucleotide coding for the polypeptide as defined herein may beachieved. In alterative a polynucleotide coding for the polypeptide asdefined herein may be partially or fully replaced with a polynucleotidesequence which does not code for a polypeptide as defined herein orwhich code for a partially or fully inactive form of a polypeptide asdefined herein. In yet another alternative one or more nucleotides canbe inserted into the polynucleotide encoding a polypeptide as definedherein resulting in the disruption of said polynucleotide and consequentpartial or full inactivation of the polypeptide as defined herein codedby the disrupted polynucleotide.

In one embodiment the mutant microbial host cell according to theinvention comprises a modification in its genome selected from

-   -   a) a full or partial deletion of a polynucleotide as defined        herein,    -   b) a full or partial replacement of a polynucleotide as defined        herein with a polynucleotide sequence which does not code for a        polypeptide as defined herein or which code for a partially or        fully inactive form of a polypeptide as defined herein    -   c) a disruption of a polynucleotide as defined herein by the        insertion of one or more nucleotides in the polynucleotide        sequence and consequent partial or full inactivation of the        polypeptide as defined herein coded by the disrupted        polynucleotide.

This modification may for example be in a coding sequence or aregulatory element required for the transcription or translation of thepolynucleotide as described above. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of a start codon or a change or a frame-shift of the openreading frame of a coding sequence. The modification of a codingsequence or a regulatory element thereof may be accomplished bysite-directed or random mutagenesis, DNA shuffling methods, DNAreassembly methods, gene synthesis (see for example Young and Dong,(2004), Nucleic Acids Research 32, (7) electronic accessnar.oupjournals.org/cgi/reprint/32/7/e59 or Gupta et al. (1968), Proc.Natl. Acad. Sci USA, 60: 1338-1344; Scarpulla et al. (1982), Anal.Biochem. 121: 356-365; Stemmer et al. (1995), Gene 164: 49-53), or PCRgenerated mutagenesis in accordance with methods known in the art.Examples of random mutagenesis procedures are well known in the art,such as for example chemical (NTG for example) mutagenesis or physical(UV for example) mutagenesis. Examples of site-directed mutagenesisprocedures are the QuickChange™ site-directed mutagenesis kit(Stratagene Cloning Systems, La Jolla, Calif.), the ‘The Altered Sites®II in vitro Mutagenesis Systems’ (Promega Corporation) or by overlapextension using PCR as described in Gene. 1989 Apr. 15; 77(1):51-9. (HoS N, Hunt H D, Horton R M, Pullen J K, Pease L R “Site-directedmutagenesis by overlap extension using the polymerase chain reaction”)or using PCR as described in Molecular Biology: Current Innovations andFuture Trends. (Eds. A. M. Griffin and H. G. Griffin. ISBN1-898486-01-8; 1995, PO Box 1, Wymondham, Norfolk, U.K.).

Preferred methods of modification are based on recombinant geneticmanipulation techniques such as partial or complete gene replacement orpartial or complete gene deletion.

For example, in case of replacement of a polynucleotide, nucleic acidconstruct or expression cassette, an appropriate DNA sequence may beintroduced at the target locus to be replaced. The appropriate DNAsequence is preferably present on a cloning vector. Preferredintegrative cloning vectors comprise a DNA fragment, which is homologousto the polynucleotide and/or has homology to the polynucleotidesflanking the locus to be replaced for targeting the integration of thecloning vector to this pre-determined locus. In order to promotetargeted integration, the cloning vector is preferably linearized priorto transformation of the cell. Preferably, linearization is performedsuch that at least one but preferably either end of the cloning vectoris flanked by sequences homologous to the DNA sequence (or flankingsequences) to be replaced. This process is called homologousrecombination and this technique may also be used in order to achieve(partial) gene deletion.

For example, a polynucleotide corresponding to the endogenouspolynucleotide may be replaced by a defective polynucleotide, that is apolynucleotide that fails to produce a (fully functional) polypeptide.By homologous recombination, the defective polynucleotide replaces theendogenous polynucleotide. It may be desirable that the defectivepolynucleotide also encodes a marker, which may be used for selection oftransformants in which the nucleic acid sequence has been modified.

Alternatively or in combination with other mentioned techniques, atechnique based on in vivo recombination of cosmids in E. coli can beused, as described in: A rapid method for efficient gene replacement inthe filamentous fungus Aspergillus nidulans (2000) Chaveroche, M-K.,Ghico, J-M. and d'Enfert C; Nucleic acids Research, vol 28, no 22.

Alternatively, modification, wherein said host cell produces less of orno protein such as the polypeptide as defined herein and encoded by apolynucleotide as described herein, may be performed by establishedanti-sense techniques using a nucleotide sequence complementary to thenucleic acid sequence of the polynucleotide. More specifically,expression of the polynucleotide by a host cell may be reduced oreliminated by introducing a nucleotide sequence complementary to thenucleic acid sequence of the polynucleotide, which may be transcribed inthe cell and is capable of hybridizing to the mRNA produced in the cell.Under conditions allowing the complementary anti-sense nucleotidesequence to hybridize to the mRNA, the amount of protein translated isthus reduced or eliminated. An example of expressing an antisense-RNA isshown in Appl. Environ. Microbiol. 2000 February; 66(2):775-82.(Characterization of a foldase, protein disulfide isomerase A, in theprotein secretory pathway of Aspergillus niger. Ngiam C, Jeenes D J,Punt P J, Van Den Hondel C A, Archer D B) or (Zrenner R, Willmitzer L,Sonnewald U. Analysis of the expression of potatouridinediphosphate-glucose pyrophosphorylase and its inhibition byantisense RNA. Planta. (1993); 190(2):247-52.).

In one embodiment the mutant microbial host cell according to theinvention is a mutant microbial host cell wherein the modification whichresults in a reduced or no production of a polypeptide as defined hereinis due to a reduced production of the mRNA encoding said polypeptide ifcompared with a parent microbial host cell which has not been modifiedand measured under the same conditions.

A modification which results in a reduced amount of the mRNA transcribedfrom the polynucleotide encoding for the polypeptide as described hereinmay be obtained via the RNA interference (RNAi) technique (FEMS Microb.Lett. 237 (2004): 317-324). In this method identical sense and antisenseparts of the nucleotide sequence, which expression is to be affected,are cloned behind each other with a nucleotide spacer in between, andinserted into an expression vector. After such a molecule istranscribed, formation of small nucleotide fragments will lead to atargeted degradation of the mRNA, which is to be affected. Theelimination of the specific mRNA can be to various extents. The RNAinterference techniques described in WO2008/053019, WO2005/05672A1,WO2005/026356A1, Oliveira et al., “Efficient cloning system forconstruction of gene silencing vectors in Aspergillus niger” (2008)Appl. Microbiol. and Biotechnol. 80 (5): 917-924 and/or Barnes et al.,“siRNA as a molecular tool for use in Aspergillus niger” (2008)Biotechnology Letters 30 (5): 885-890 may be used at this purpose.

A modification which results in a polypeptide with decreased or noenzymatic activity as defined herein can be obtained by differentmethods, for example by an antibody directed against such a polypeptideor a chemical inhibitor or a protein inhibitor or a physical inhibitor(Tour O. et al, (2003) Nat. Biotech: Genetically targetedchromophore-assisted light inactivation. Vol. 21. no. 12:1505-1508) orpeptide inhibitor or an anti-sense molecule or RNAi molecule (R. S.Kamath_et al, (2003) Nature: Systematic functional analysis of theCaenorhabditis elegans genome using RNAi.vol. 421, 231-237).

In addition of the above-mentioned techniques or as an alternative, itis also possible to inhibiting the activity of a polypeptide as definedherein, or to re-localize the polypeptide as defined herein by means ofalternative signal sequences (Ramon de Lucas, J., Martinez O, Perez P.,Isabel Lopez, M., Valenciano, S. and Laborda, F. The Aspergillusnidulans carnitine carrier encoded by the acuH gene is exclusivelylocated in the mitochondria. FEMS Microbiol Lett. 2001 Jul. 24;201(2):193-8.) or retention signals (Derkx, P. M. and Madrid, S. M. Thefoldase CYPB is a component of the secretory pathway of Aspergillusniger and contains the endoplasmic reticulum retention signal HEEL. Mol.Genet. Genomics. 2001 December; 266(4):537-545.), or by targeting thepolypeptide to a peroxisome which is capable of fusing with amembrane-structure of the cell involved in the secretory pathway of thecell, leading to secretion outside the cell of the polypeptide (e.g. asdescribed in WO2006/040340).

Alternatively or in combination with above-mentioned techniques,inhibition of polypeptide enzymatic activity as defined herein can alsobe obtained, e.g. by UV or chemical mutagenesis (Mattern, I. E., vanNoort J. M., van den Berg, P., Archer, D. B., Roberts, I. N. and van denHondel, C. A., Isolation and characterization of mutants of Aspergillusniger deficient in extracellular proteases. Mol Gen Genet. 1992 August;234(2):332-6.) or by the use of inhibitors inhibiting enzymatic activityof a polypeptide as described herein (e.g. nojirimycin, which functionas inhibitor for β-glucosidases (Carrel F. L. Y. and Canevascini G.Canadian Journal of Microbiology (1991) 37(6): 459-464; Reese E. T.,Parrish F. W. and Ettlinger M. Carbohydrate Research (1971) 381-388)).

In an embodiment according to the invention the modification in thegenome of the mutant microbial host cell according to the invention is amodification in at least one position of a polynucleotide as definedabove encoding for the polypeptide, as defined above.

In the context of the present invention the “parent microbial host cell”and the “mutant microbial host cell” may be any type of host cell. Thespecific embodiments of the mutant microbial host cell are hereafterdescribed. It will be clear to those skilled in the art that embodimentsapplicable to the mutant microbial host cell are as well applicable tothe parent microbial host cell unless otherwise indicated.

The mutant microbial host cell according to the present invention may bea prokaryotic cell. Preferably, the prokaryotic host cell is bacterialcell. The term “bacterial cell” includes both Gram-negative andGram-positive microorganisms. Suitable bacteria may be selected frome.g. Escherichia, Anabaena, Caulobactert, Gluconobacter, Rhodobacter,Pseudomonas, Paracoccus, Bacillus, Brevibacterium, Corynebacterium,Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter,Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus orStreptomyces. Preferably, the bacterial cell is selected from the groupconsisting of B. subtilis, B. amyloliquefaciens, B. licheniformis, B.puntis, B. megaterium, B. halodurans, B. pumilus, G. oxydans,Caulobactert crescentus CB 15, Methylobacterium extorquens, Rhodobactersphaeroides, Pseudomonas zeaxanthinifaciens, Paracoccus denitrificans,E. coli, C. glutamicum, Staphylococcus carnosus, Streptomyces lividans,Sinorhizobium melioti and Rhizobium radiobacter.

According to an embodiment, the mutant microbial host cell according tothe invention is a eukaryotic host cell. Preferably, the eukaryotic cellis a mammalian, insect, plant, fungal, or algal cell. Preferredmammalian cells include e.g. Chinese hamster ovary (CHO) cells, COScells, 293 cells, PerC6 cells, and hybridomas. Preferred insect cellsinclude e.g. Sf9 and Sf21 cells and derivatives thereof. Morepreferably, the eukaryotic cell is a fungal cell, i.e. a yeast cell,such as Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia strain. More preferably fromKluyveromyces lactis, S. cerevisiae, Hansenula polymorpha, Yarrowialipolytica and Pichia pastoris, or a filamentous fungal cell. Mostpreferably, the eukaryotic cell is a filamentous fungal cell.

Filamentous fungi include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium,Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus,Schizophyllum, Talaromyces, Rasamsonia, Thermoascus, Thielavia,Tolypocladium, and Trichoderma.

Preferred filamentous fungal cells belong to a species of an Acremonium,Aspergillus, Chrysosporium, Myceliophthora, Penicillium, Talaromyces,Rasamsonia, Thielavia, Fusarium or Trichoderma genus, and mostpreferably a species of Aspergillus niger, Acremonium alabamense,Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae,Aspergillus fumigatus, Talaromyces emersonfi, Rasamsonia emersonii,Aspergillus oryzae, Chrysosporium lucknowense, Fusarium oxysporum,Myceliophthora thermophila, Trichoderma reesei, Thielavia terrestris orPenicillium chrysogenum. A more preferred host cell belongs to the genusAspergillus, more preferably the host cell belongs to the speciesAspergillus niger. When the host cell according to the invention is anAspergillus niger host cell, the host cell preferably is CBS 513.88,CBS124.903 or a derivative thereof.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS),Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL), and All-Russian Collection ofMicroorganisms of Russian Academy of Sciences, (abbreviation inRussian—VKM, abbreviation in English—RCM), Moscow, Russia. Usefulstrains in the context of the present invention may be Aspergillus nigerCBS 513.88, CBS124.903, Aspergillus oryzae ATCC 20423, IFO 4177, ATCC1011, CBS205.89, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, P. chrysogenum Wisconsin54-1255 (ATCC28089),Penicillium citrinum ATCC 38065, Penicillium chrysogenum P2, Thielaviaterrestris NRRL8126, Talaromyces emersonfi CBS 124.902, Acremoniumchrysogenum ATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 orATCC 56765 or ATCC 26921, Aspergillus sojae ATCC11906, Myceliophthorathermophila C1, Garg 27K, VKM-F 3500 D, Chrysosporium lucknowense C1,Garg 27K, VKM-F 3500 D, ATCC44006 and derivatives thereof.

According to one embodiment of the invention, when the mutant microbialhost cell according to the invention is a filamentous fungal host cell,the mutant microbial host cell may further comprise one or moremodifications in its genome such that the mutant microbial host cell isdeficient in the production of at least one product selected fromglucoamylase (glaA), acid stable alpha-amylase (amyA), neutralalpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin,preferably ochratoxin and/or fumonisin, a protease transcriptionalregulator prtT, PepA, a product encoded by the gene hdfA and/or hdfB, anon-ribosomal peptide synthase npsE if compared to a parent host celland measured under the same conditions.

Oxalic acid hydrolase (oahA) is a component of the synthesis pathway ofoxalic acid in many host cells. A host cell deficient in oahA will bedeficient in oxalic acid. Oxalic acid is an unwanted by-product in manyapplications such as food-applications. Furthermore, oxalic acid lowersthe pH of the medium cultivations of host cell producing this component,resulting in lowered yields; i.e. yield is increased in oxalic aciddeficient host cells. It is therefore advantageous if the microbial hostcell according to the invention is deficient in oahA. OahA deficienthost cells and preferred methods of producing said host cells areextensively described in WO 2000/50576 and WO2004/070022. A preferredmethod to produce an oahA deficient host cell is the recombinant methodof disruption described in WO 2000/50576. Preferably, the mutantmicrobial host cell according to the invention is deficient in oahA.Preferably, the oahA is a fungal oahA. More preferably, the oahA is theoahA from Aspergillus. Even more preferably the oahA is the oahA fromAspergillus niger. Even more preferably the oahA is the oahA fromAspergillus niger CBS 513.88. Most preferably, the oahA comprises thesequence of An10g00820.

prtT is a transcriptional activator of proteases in eukaryotic cells.Several fungal transcriptional activators of proteases have beenrecently described in WO 00/20596, WO 01/68864, WO 2006/040312 and WO2007/062936. These transcriptional activators were isolated fromAspergillus niger (A. niger), Aspergillus fumigatus (A. fumigatus),Penicillium chrysogenum (P. chrysogenum) and Aspergillus oryzae (A.oryzae). These transcriptional activators of protease genes can be usedto improve a method for producing a polypeptide in a fungal cell,wherein the polypeptide is sensitive for protease degradation. When themicrobial host cell according to the invention is deficient in prtT, thehost cell will produce less proteases that are under transcriptionalcontrol of prtT. It is therefore advantageous when the host cellaccording to the invention is deficient in prtT. prtT deficient hostsand preferred methods to produce these hosts are extensively describedin WO 01/68864, WO 2006/040312. WO 01/68864 and WO 2006/040312 describerecombinant and classic methods to disrupt the prtT coding sequence. WO2007/062936 describes disruption of the prtT binding site in a proteasepromoter. Disruption of the binding site impedes binding of prtT to thebinding site. Consequently, the transcription of the protease is notactivated by prtT and less protease is produced.

Preferably, the mutant microbial host cell according to the inventioncomprises a polynucleotide encoding prtT, said polynucleotide comprisinga modification, wherein the host cell is deficient in the production ofprtT compared to a parent cell it originates from when cultivated undercomparable conditions. Preferably, the prtT is a fungal prtT. Morepreferably, the prtT is the prtT from Aspergillus. Even more preferablythe prtT is the prtT from Aspergillus niger. Even more preferably theprtT is the prtT from Aspergillus niger CBS 513.88. Most preferably, theprtT comprises the sequence of An04g06940.

The term “glucoamylase” (glaA) is identical to the term“amyloglucosidase” and is defined herein as an enzyme having dextrin6-alpha-D-glucanohydrolase activity which catalyses the endo hydrolysisof 1, 6-alpha-D-glucoside linkages at points of branching in chains of1, 4-linked alpha-D-glucose residues and terminal 1, 4-linkedalpha-D-glucose residues. Glucoamylase activity can be measured asAGIU/ml by determining the liberation of paranitrofenol from thesubstrate p-nitrophenyl-α-D-glucopyranoside (Sigma). This results in ayellow colour, whose absorbance can be measured at 405 nm using aspectrophotometer. 1 AGIU is the quantity of enzyme, which produces 1μmole of glucose per minute at pH 4.3 and 60° C. from a soluble starchsubstrate. In WO98/46772 additional details of the assay can be found.

Preferably, the mutant microbial host cell according to the inventioncomprises a polynucleotide encoding glaA, said polynucleotide comprisinga modification, wherein the host cell is deficient in the production ofglaA compared to a parent cell it originates from when cultivated undercomparable conditions. Preferably, the glaA is a fungal glaA. Morepreferably, the glaA is the glaA from Aspergillus. Even more preferablythe glaA is the glaA from Aspergillus niger. Even more preferably theglaA is the glaA from Aspergillus niger CBS 513.88. Most preferably, theglaA comprises the sequence of An03g06550.

The term “alpha-amylase” is defined herein as 1, 4-alpha-D-glucanglucanohydrolase activity which catalyzes the endohydrolysis ofpolysaccharides with three or more alpha-1, 4-linked glucose units inthe presence of water to malto-oligosaccharides. To determine the(neutral) alpha-amylase activity, the Megazyme cereal alpha-amylase kitis used (Megazyme, CERALPHA alpha amylase assay kit, catalogus. ref.K-CERA, year 2000-2001), according a protocol of the supplier. Themeasured activity is based on hydrolysis of non-reducing-endblockedρ-nitrophenyl maltoheptaoside in the presence of excess glucoamylase andα-glucosidase at a pH of 7.0. The amount of formed ρ-nitrophenol is ameasure for alpha-amylase activity present in a sample.

The term “acid stable alpha-amylase” (amyA) is defined herein as anenzyme having alpha-amylase activity with optimal activity in the acidpH range. To determine the acid stable alpha-amylase activity, also theMegazyme cereal alpha-amylase kit is used (Megazyme, CERALPHA alphaamylase assay kit, catalogus. ref. K-CERA, year 2000-2001), according aprotocol of the supplier but at an acid pH. The measured activity isbased on hydrolysis of non-reducing-endblocked ρ-nitrophenylmaltoheptaoside in the presence of excess glucoamylase and α-glucosidaseat a pH of 4.5. The amount of formed ρ-nitrophenol is a measure for acidstable alpha-amylase activity present in a sample.

Preferably, the host cell according to the invention comprises apolynucleotide encoding AmyA, said polynucleotide comprising amodification, wherein the host cell is deficient in amyA compared to theparent cell it originates from when cultivated under comparableconditions. Preferably, the amyA is a fungal amyA. More preferably, theamyA is the amyA from Aspergillus. Even more preferably the amyA is theamyA from Aspergillus niger. Even more preferably the amyA is the amyAfrom Aspergillus niger CBS 513.88. Most preferably, the amyA comprisesthe sequence of An11g03340.

The term “neutral alpha-amylase activity” (amy) is defined herein as anenzyme having alpha-amylase activity with optimal activity in theneutral pH range.

Preferably, the host cell according to the invention comprises apolynucleotide encoding AmyB, said polynucleotide comprising amodification, wherein the host cell is deficient in amyBI and/or amyBIIcompared to the parent cell it originates from when cultivated undercomparable conditions. More preferably, the microbiaol host cellaccording to the invention is deficient in amyBI and amy BII.Preferably, the amyB a is a fungal amyB. More preferably, the amyB isthe amyB from Aspergillus. Even more preferably the amyB is the amyBIfrom Aspergillus niger. Even more preferably the amyB is the amyBI fromAspergillus niger CBS 513.88. Most preferably, the amyBI comprises thesequence of An12g06930. Even more preferably the amyB is the amyBII fromAspergillus niger. Even more preferably the amyB is the amyBII fromAspergillus niger CBS 513.88. Most preferably, the amyBII comprises thesequence of An05g02100.

The term toxin associated polynucleotide is defined herein as a genecluster, a multitude of genes, a gene or part thereof encoding acompound, or biochemical pathway responsible for the biosynthesis orsecretion of at least one toxin or toxin intermediate compound. Saidcompound may e.g. be a polypeptide, which may be an enzyme.

A number of host cells, especially fungi, which are used as host cellsin the production of polypeptides of interest possesses genes encodingenzymes involved in the biosynthesis of various toxins. For example,cyclopiazonic acid, kojic acid, 3-nitropropionic acid and aflatoxins areknown toxins, which are formed in, e.g., Aspergillus flavus. Similarly,trichothecenes are formed in a number of fungi, e.g., in Fusarium sp.such as Fusarium venenatum and in Trichoderma and ochratoxin may beproduced by Aspergillus. Recently, sequencing of the genome of anindustrial Aspergillus niger host strain revealed a fumonisin genecluster (Pel et al., “Genome sequencing and analysis of the versatilecell factory Aspergillus niger CBS 513.88”. Nat Biotechnol. 2007February; 25 (2):221-231). The formation of such toxins during thefermentation of compounds of interest is highly undesirable as thesetoxins may present a health hazard to operators, customers and theenvironment. Consequently, a toxin deficient host cell enablestoxin-free production of a compound of interest. The toxin-free compoundis easier to produce since no toxin has to be removed from the product.Furthermore, the regulatory approval procedure for the compound iseasier.

Preferably, the mutant microbial host cell according to the inventioncomprises a toxin associated polynucleotide encoding a compound (whichmay e.g. be a polypeptide which may be an enzyme) or biochemicalpathway, said toxin associated polynucleotide comprising a modification,wherein the host cell is deficient in the production of said toxin or atoxin intermediate compound compared to the parent cell it originatesfrom when cultivated under comparable conditions. Preferably, the toxinor toxin intermediate compound is a fungal toxin or toxin intermediatecompound. More preferably, the toxin or toxin intermediate compound is atoxin or toxin intermediate compound from Aspergillus. Even morepreferably the toxin or the toxin intermediate compound is a toxin ortoxin intermediate compound from Aspergillus niger. Even more preferablythe toxin or toxin intermediate compound is a toxin or toxinintermediate compound from Aspergillus niger CBS 513.88. Even morepreferably, the toxin or the toxin intermediate compound is fumonisin ora fumonisin intermediate compound. Even more preferably, the toxin orthe toxin intermediate compound is ochratoxin or an ochratoxinintermediate compound. Most preferably, the toxin or the toxinintermediate compound is ochratoxin or fumonisin or an ochratoxin or afumonisin intermediate compound.

Preferably, the toxin associated polynucleotide encodes a compound(which may e.g. be a polypeptide which may be an enzyme) or abiochemical pathway which is involved in the production of a fungaltoxin or toxin intermediate compound. More preferably, a toxin or toxinintermediate compound from Aspergillus. Even more preferably, a toxin ortoxin intermediate compound from Aspergillus niger. Even morepreferably, a toxin or toxin intermediate compound from Aspergillusniger CBS 513.88. Even more preferably, a fumonisin or a fumonisinintermediate compound. Even more preferably, a fumonisin-B or afumonisin-B intermediate compound. Even more preferably, a fumonisin-B2or a fumonisin-B2 intermediate compound. Even more preferably, the toxinassociated polynucleotide comprises the sequence of the fumonisincluster from An01g06820 until An01g06930. Most preferably, the toxinassociated polynucleotide comprises the sequence of An01g06930.

In another preferred embodiment, the toxin associated polynucleotideencodes a compound (which may e.g. be a polypeptide which may be anenzyme) or a biochemical pathway which is involved in ochratoxin or anochratoxin intermediate compound. More preferably, an ochratoxin A or anochratoxin A intermediate compound. More preferably, the toxinassociated polynucleotide comprises the sequence of the cluster fromAn15g07880 until An15g07930. Most preferably, the toxin associatedpolynucleotide comprises the sequence of An15g07910 and/or the sequenceof An15g07920.

Preferably, the mutant microbial host cell according to the inventioncomprises at least one toxin associated polynucleotide encoding acompound (which may e.g. be a polypeptide which may be an enzyme) orbiochemical pathway, said toxin associated polynucleotide comprising atleast one modification, wherein the host cell is deficient in theproduction of a toxin or, toxin intermediate compound compared to theparent cell it originates from when cultivated under comparableconditions.

More preferably, the host cell according to the invention comprises twotoxin associated polynucleotides, said two toxin associatedpolynucleotides each comprising at least one modification, wherein thehost cell is preferably deficient in the production of fumonisin andochratoxin compared to the parent cell it originates from whencultivated under comparable conditions.

Even more preferably, the mutant microbial host cell according to theinvention comprises three or more toxin associated polynucleotides saidthree or more toxin associated polynucleotides each comprising at leastone modification, wherein the host cell is preferably deficient in theproduction of fumonisin, ochratoxin and at least one additional toxin ortoxin intermediate compound compared to the parent cell it originatesfrom when cultivated under comparable conditions.

Therefore, when the mutant microbial host cell according to theinvention is a filamentous fungal host cell the host cell may compriseone or more modifications in its genome to result in a deficiency in theproduction of the major extracellular aspartic protease PepA. Forexample the host cell according to the invention may further comprise adisruption of the pepA gene encoding the major extracellular asparticprotease PepA. More preferably, the pepA is the pepA from Aspergillus.Even more preferably the pepA is the pepA from Aspergillus niger. Evenmore preferably the pepA is the pepA from Aspergillus niger CBS 513.88.Most preferably, the pepA comprises the sequence of An14g04710.

Preferably, the efficiency of targeted integration of a polynucleotideto a pre-determined site into the genome of the mutant microbial hostcell according to the invention is increased by making the celldeficient in a component in NHR (non-homologous recombination).Preferably, the mutant microbial host cell according to the inventioncomprises a polynucleotide encoding an NHR component comprising amodification, wherein said host cell is deficient in the production ofsaid NHR component compared to a parent cell it originates from whencultivated under the same conditions.

The NHR component to be modified can be any NHR component known to theperson skilled in the art. Preferred NHR components to be modified areselected from the group of filamentous fungal homologues of yeast KU70,KU80, MRE11, RAD50, RAD51, RAD52, XRS2, SIR4, LIG4. More preferred NHRcomponents to be modified are filamentous fungal homologues of yeastKU70 and KU80, preferably hdfA (homologue of yeast KU70) or homologuesthereof and hdfB (homologue of yeast KU80) or homologues thereof. Themost preferred NHR component to be modified is KU70 or hdfA, or ahomologue thereof. Another preferred NHR component to be modified isKU80 or hdfB, or a homologue thereof. Methods to obtain such host celldeficient in a component involved in NHR are known to the skilled personand are extensively described in WO2005/095624. Preferably the hdfA geneis the hdfA gene from A. niger, more preferably the hdfA from A. nigeraccording to SEQ ID NO: 1 of WO2005/095624. In another preferredembodiment the hdfB gene is the hdfB gene from A. niger, more preferablythe hdfB from A. niger according to SEQ ID NO: 4 of WO2005/095624.

Therefore when the mutant microbial host cell according to the inventionis a filamentous fungal host cell the host cell according to theinvention may additionally comprises one or more modifications in itsgenome to result in a deficiency in the production of the productencoded by the hdf A gene (as depicted in SEQ ID NO: 3 of WO2005/095624) and/or hdfB gene (as depicted in SEQ ID NO: 6 of WO2005/095624). For example the host cell according to the invention mayfurther comprise a disruption of the hdfA and/or hdfB gene. Filamentousfungal host cells which are deficient in a product encoded by the hdfAand/or hdfB gene have been described in WO 2005/095624.

When the mutant microbial host cell according to the invention is afilamentous fungal host cell the host cell according to the inventionmay additionally comprise a modification in its genome which results inthe deficiency in the production of the non-ribosomal peptide synthasenpsE. Such host cells deficient in the production of non-ribosomalpeptide synthase npsE have been described in WO2012/001169 (npsE has agenomic sequence as depicted in SEQ ID NO: 35, a coding sequencedepicted in SEQ ID NO: 36, the mRNA depicted in SEQ ID NO: 37 and thenrps protein depicted in SEQ ID NO: 38 of WO2012/001169).

The mutant microbial host cell according to the invention mayadditionally comprise a modification in its genome which results in thedeficiency in the production of the α-amylase amyC. Such host cellsdeficient in the production of the α-amylase amyC have been described ina co-pending International patent application filed on 19 Jul. 2013entitled “Amylase-Deficient Strain” and which claims priority fromEP12177173.7, U.S. 61/673,589, EP12177171.1 and U.S. 61/673,607 allfiled on 19 Jul. 2012. amyC has a genomic sequence as depicted in SEQ IDNO: 1 or 5 and a coding sequence depicted in SEQ ID NO: 2 or 6 and theAmyC protein as depicted in SEQ ID NO: 3 or 7 with the mature AmyCprotein shown in SEQ ID NO: 4 and 8 of this co-pending Internationalpatent application) SEQ ID NOs: 1 and 5 of the co-pending applicationcorrespond to SEQ ID NO: 13 herein. SEQ ID NOs: 2 and 6 of theco-pending application correspond to SEQ ID NOs: 14 and 17 hereinrespectively. SEQ ID NOs: 3 and 7 of the co-pending applicationcorrespond to SEQ ID NOs: 15 and 18 herein respectively. SEQ ID NOs: 4and 8 of the co-pending application correspond to SEQ ID NOs: 16 and 19herein respectively.

The deficiency in the production of at least one product selected fromglucoamylase (glaA), acid stable alpha-amylase (amyA), neutralalpha-amylase (amyBI and amyBII), oxalic acid hydrolase (oahA), a toxin,preferably ochratoxin and/or fumonisin, a protease transcriptionalregulator prtT, PepA, a product encoded by the gene hdfA and/or hdfB, anon-ribosomal peptide synthase npsE, amylase amyC if compared to aparent host cell and measured under the same conditions may already bepresent in the parent host cell from which the mutant microbial hostcell according to the invention is derived.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA and optionallyat least another product selected from the group consisting of acidstable alpha-amylase (amyA), neutral alpha-amylase (amyBI and amyBII),oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/orfumonisin, a protease transcriptional regulator prtT, PepA, a productencoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthasenpsE, amylase amyC if compared to a parent host cell and measured underthe same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA andoptionally at least another product selected from the group consistingof acid stable alpha-amylase (amyA), neutral alpha-amylase (amyBI andamyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxinand/or fumonisin, a protease transcriptional regulator prtT, a productencoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthasenpsE, amylase amyC if compared to a parent host cell and measured underthe same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA) and optionally at least another productselected from the group consisting of neutral alpha-amylase (amyBI andamyBII), oxalic acid hydrolase (oahA), a toxin, preferably ochratoxinand/or fumonisin, a protease transcriptional regulator prtT, a productencoded by the gene hdfA and/or hdfB, a non-ribosomal peptide synthasenpsE, amylase amyC if compared to a parent host cell and measured underthe same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and optionallyat least another product selected from the group consisting of neutralalpha-amylase amyBII, oxalic acid hydrolase (oahA), a toxin, preferablyochratoxin and/or fumonisin, a protease transcriptional regulator prtT,a product encoded by the gene hdfA and/or hdfB, a non-ribosomal peptidesynthase npsE, amylase amyC if compared to a parent host cell andmeasured under the same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, andoptionally at least another product selected from the group consistingof oxalic acid hydrolase (oahA), a toxin, preferably ochratoxin and/orfumonisin, a protease transcriptional regulator prtT, a product encodedby the gene hdfA and/or hdfB, a non-ribosomal peptide synthase npsE,amylase amyC if compared to a parent host cell and measured under thesame conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, aproduct encoded by the gene hdfA and optionally at least another productselected from the group consisting of oxalic acid hydrolase (oahA), atoxin, preferably ochratoxin and/or fumonisin, a proteasetranscriptional regulator prtT, a product encoded by the gene hdfB, anon-ribosomal peptide synthase npsE, amylase amyC if compared to aparent host cell and measured under the same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, aproduct encoded by the gene hdfA, oxalic acid hydrolase (oahA) andoptionally at least another product selected from the group consistingof, a toxin, preferably ochratoxin and/or fumonisin, a proteasetranscriptional regulator prtT, a product encoded by the gene hdfB, anon-ribosomal peptide synthase npsE, amylase amyC if compared to aparent host cell and measured under the same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, aproduct encoded by the gene hdfA, oxalic acid hydrolase (oahA),ochratoxin, fumonisin, and optionally at least another product selectedfrom the group consisting of a protease transcriptional regulator prtT,a product encoded by the gene hdfB, a non-ribosomal peptide synthasenpsE, amylase amyC if compared to a parent host cell and measured underthe same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, aproduct encoded by the gene hdfA, oxalic acid hydrolase (oahA),ochratoxin, fumonisin, a protease transcriptional regulator prtT andoptionally at least another product selected from the group consistingof a product encoded by the gene hdfB, a non-ribosomal peptide synthasenpsE, amylase amyC if compared to a parent host cell and measured underthe same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, aproduct encoded by the gene hdfA, oxalic acid hydrolase (oahA),ochratoxin, fumonisin, a protease transcriptional regulator prtT, anon-ribosomal peptide synthase npsE and optionally at least anotherproduct selected from the group consisting of a product encoded by thegene hdfB, amylase amyC if compared to a parent host cell and measuredunder the same conditions.

In one embodiment the mutant microbial cell according to the inventionfurther comprises a deficiency in the production of glaA, PepA, acidstable alpha-amylase (amyA), neutral alpha-amylase amyBI and amyBII, aproduct encoded by the gene hdfA, oxalic acid hydrolase (oahA),ochratoxin, fumonisin, a protease transcriptional regulator prtT,amylase amyC and optionally at least another product selected from thegroup consisting of a product encoded by the gene hdfB, a non-ribosomalpeptide synthase npsE, if compared to a parent host cell and measuredunder the same conditions.

In a more preferred embodiment the mutant microbial cell according tothe invention further has a reduced amylase background and comprises adeficiency in the production of glaA, acid stable alpha-amylase (amyA),neutral alpha-amylase amyBI and amyBII, if compared to a parent hostcell and measured under the same conditions. Such a microbial mutantcell may also comprise a deficiency in the production of a filamentousfungal homolog of KU70 or KU80. Such a microbial mutant cell may alsocomprise a deficiency in the production of a toxin. Such a microbialmutant cell may also comprise a deficiency in the production of afilamentous fungal homolog of KU70 or KU80 and a deficiency in theproduction of a toxin.

In an even more preferred embodiment the mutant microbial cell accordingto the invention has a reduced amylase background and further comprisesa deficiency in the production of glaA, acid stable alpha-amylase(amyA), neutral alpha-amylase amyBI, amyBII and amyC if compared to aparent host cell and measured under the same conditions. Such amicrobial mutant cell may also comprise a filamentous fungal homolog ofKU70 or KU80. Such a microbial mutant cell may also comprise adeficiency in the production of a toxin. Such a microbial mutant cellmay also comprise a deficiency in the production of a filamentous fungalhomolog of KU70 or KU80 and a deficiency in the production of a toxin.

In a most preferred embodiment the mutant microbial cell according tothe invention further has a reduced alpha-amylase background andcomprises a deficiency in the production acid stable alpha-amylase(amyA), neutral alpha-amylase amyBI and amyBII and, optionally, amyC ifcompared to a parent host cell and measured under the same conditions.Such a microbial mutant cell may also comprise a filamentous fungalhomolog of KU70 or KU80. Such a microbial mutant cell may also comprisea deficiency in the production of a toxin. Such a microbial mutant cellmay also comprise a deficiency in the production of a filamentous fungalhomolog of KU70 or KU80 and a deficiency in the production of a toxin.

When the mutant microbial host cell according to the invention is afilamentous fungal host cell the host cell may additionally comprise atleast two substantially homologous DNA domains suitable for integrationof one or more copies of a polynucleotide encoding a compound ofinterest wherein at least one of the at least two substantiallyhomologous DNA domains is adapted to have enhanced integrationpreference for the polynucleotide encoding a compound of interestcompared to the substantially homologous DNA domain it originates from,and wherein the substantially homologous DNA domain where the adaptedsubstantially homologous DNA domain originates from has a geneconversion frequency that is at least 10% higher than one of the otherof the at least two substantially homologous DNA domains. These cellshave been described in WO2011/009700. Strains containing two or morecopies of these substantially homologous DNA domains are also referredhereafter as strain containing two or more amplicons. Examples of hostcells comprising such amplicons are e.g. described in van Dijck et al,2003, Regulatory Toxicology and Pharmacology 28; 27-35: On the safety ofa new generation of DSM Aspergillus niger enzyme production strains. Invan Dijck et al, an Aspergillus niger strain is described that comprises7 amplified glucoamylase gene loci, i.e. 7 amplicons. Preferred hostcells within this context are filamentous fungus host cells, preferablyA. niger host cells, comprising two or more amplicons, preferably two ormore ΔglaA amplicons (preferably comprising 3, 4, 5, 6, 7 ΔglaAamplicons) wherein the amplicon which has the highest frequency of geneconversion, has been adapted to have enhanced integration preference forthe polynucleotide encoding a compound of interest compared to theamplicon it originates from. Adaptation of the amplicon can be performedaccording to any one of the methods described in WO2011/009700 (which ishere fully incorporated by reference). An example of these host cells,described in WO2011/009700, are host cells comprising three ΔglaAamplicons being a BamHI truncated amplicon, a SalI truncated ampliconand a BglII truncated amplicon and wherein the BamHI amplicon has beenadapted to have enhanced integration preference for a polynucleotideencoding a compound of interest compared to the BamHI amplicon itoriginates from. Host cells comprising two or more amplicons wherein oneamplicon has been adapted to have enhanced integration preference for apolynucleotide encoding a compound of interest compared to the ampliconit originates from are hereafter referred as host cells comprising anadapted amplicon.

When the mutant microbial host cell according to the invention is afilamentous fungal host cell the host cell according to the inventionmay additionally comprises a modification of Sec61. A preferred SEC61modification is a modification which results in a one-way mutant ofSEC61; i.e. a mutant wherein the de novo synthesized protein can enterthe ER via SEC61, but the protein cannot leave the ER via SEC61. Suchmodifications are extensively described in WO2005/123763. In a preferredembodiment the mutant microbial host cell comprises a modification in aSec61 as depicted in SEQ ID NO: 3 of WO2005/123763. Most preferably, theSEC 61 modification is the S376W mutation in which Serine 376 isreplaced by Tryptophan in SEQ ID NO: 3 of WO2005/123763.

In a preferred embodiment, the mutant microbial host cell according tothe invention comprises at least one polynucleotide coding for acompound of interest or at least one polynucleotide coding for acompound involved in the production of a compound of interest by thecell.

The compound of interest can be any biological compound. The biologicalcompound may be biomass or a biopolymer or metabolite. The biologicalcompound may be encoded by a single polynucleotide or a series ofpolynucleotides composing a biosynthetic or metabolic pathway or may bethe direct result of the product of a single polynucleotide or productsof a series of polynucleotides. The biological compound may be native tothe host cell or heterologous.

The term “heterologous biological compound” is defined herein as abiological compound which is not native to the cell; or a nativebiological compound in which structural modifications have been made toalter the native biological compound.

The term “biopolymer” is defined herein as a chain (or polymer) ofidentical, similar, or dissimilar subunits (monomers). The biopolymermay be any biopolymer. The biopolymer may for example be, but is notlimited to, a nucleic acid, polyamine, polyol, polypeptide (orpolyamide), or polysaccharide.

The biopolymer may be a polypeptide. The polypeptide may be anypolypeptide having a biological activity of interest. The term“polypeptide” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. Polypeptides further include naturally occurring allelic andengineered variations of the above-mentioned polypeptides and hybridpolypeptides. The polypeptide may be native or may be heterologous tothe host cell. The polypeptide may be a collagen or gelatin, or avariant or hybrid thereof. The polypeptide may be an antibody or partsthereof, an antigen, a clotting factor, an enzyme, a hormone or ahormone variant, a receptor or parts thereof, a regulatory protein, astructural protein, a reporter, or a transport protein, protein involvedin secretion process, protein involved in folding process, chaperone,peptide amino acid transporter, glycosylation factor, transcriptionfactor, synthetic peptide or oligopeptide, intracellular protein. Theintracellular protein may be an enzyme such as, a protease, ceramidases,epoxide hydrolase, aminopeptidase, acylases, aldolase, hydroxylase,aminopeptidase, lipase. The polypeptide may also be an enzyme secretedextracellularly. Such enzymes may belong to the groups ofoxidoreductase, transferase, hydrolase, lyase, isomerase, ligase,catalase, cellulase, chitinase, cutinase, deoxyribonuclease, dextranase,esterase. The enzyme may be a carbohydrase, e.g. cellulases such asendoglucanases, β-glucanases, cellobiohydrolases or β-glucosidases,hemicellulases or pectinolytic enzymes such as xylanases, xylosidases,mannanases, galactanases, galactosidases, pectin methyl esterases,pectin lyases, pectate lyases, endo polygalacturonases,exopolygalacturonases rhamnogalacturonases, arabanases,arabinofuranosidases, arabinoxylan hydrolases, galacturonases, lyases,or amylolytic enzymes; hydrolase, isomerase, or ligase, phosphatasessuch as phytases, esterases such as lipases, proteolytic enzymes,oxidoreductases such as oxidases, transferases, or isomerases. Theenzyme may be a phytase. The enzyme may be an aminopeptidase,asparaginase, amylase, a maltogenic amylase, carbohydrase,carboxypeptidase, endo-protease, metallo-protease, serine-proteasecatalase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase,protein deaminase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phospholipase, galactolipase,chlorophyllase, polyphenoloxidase, ribonuclease, transglutaminase, orglucose oxidase, hexose oxidase, monooxygenase.

Preferably the compound of interest is a heterologous product.Preferably the compound of interest is a glucose oxidase. Morepreferably the compound of interest is a heterologous glucose oxidase.In another preferred embodiment the compound of interest is a lipolyticenzyme, e.g. a lipolytic enzyme having one or more of the activitiesselected from the group consisting of: lipase (triacyl glycerol lipase),phospholipase (e.g phospholipase A1 and/or phospholipase A2 and/orphospholipase B and/or phospholipase C), galactolipase.

According to the present invention, a polypeptide or enzyme also can bea product as described in WO2010/102982. According to the presentinvention, a polypeptide can also be a fused or hybrid polypeptide towhich another polypeptide is fused at the N-terminus or the C-terminusof the polypeptide or fragment thereof. A fused polypeptide is producedby fusing a nucleic acid sequence (or a portion thereof) encoding onepolypeptide to a nucleic acid sequence (or a portion thereof) encodinganother polypeptide.

Techniques for producing fusion polypeptides are known in the art, andinclude, ligating the coding sequences encoding the polypeptides so thatthey are in frame and expression of the fused polypeptide is undercontrol of the same promoter (s) and terminator. The hybrid polypeptidesmay comprise a combination of partial or complete polypeptide sequencesobtained from at least two different polypeptides wherein one or moremay be heterologous to the host cell. Example of fusion polypeptides andsignal sequence fusions are for example as described in WO2010/121933.

The biopolymer may be a polysaccharide. The polysaccharide may be anypolysaccharide, including, but not limited to, a mucopolysaccharide(e.g., heparin and hyaluronic acid) and nitrogen-containingpolysaccharide (e.g., chitin). In a more preferred option, thepolysaccharide is hyaluronic acid.

The polynucleotide coding for the compound of interest or coding for acompound involved in the production of the compound of interestaccording to the invention may encode an enzyme involved in thesynthesis of a primary or secondary metabolite, such as organic acids,carotenoids, (beta-lactam) antibiotics, and vitamins. Such metabolitemay be considered as a biological compound according to the presentinvention.

The term “metabolite” encompasses both primary and secondarymetabolites; the metabolite may be any metabolite. Preferred metabolitesare citric acid, gluconic acid, adipic acid, fumaric acid, itaconic acidand succinic acid.

The metabolite may be encoded by one or more genes, such as in abiosynthetic or metabolic pathway. Primary metabolites are products ofprimary or general metabolism of a cell, which are concerned with energymetabolism, growth, and structure. Secondary metabolites are products ofsecondary metabolism (see, for example, R. B. Herbert, The Biosynthesisof Secondary Metabolites, Chapman and Hall, New York, 1981).

The primary metabolite may be, but is not limited to, an amino acid,fatty acid, nucleoside, nucleotide, sugar, triglyceride, or vitamin.

The secondary metabolite may be, but is not limited to, an alkaloid,coumarin, flavonoid, polyketide, quinine, steroid, peptide, or terpene.The secondary metabolite may be an antibiotic, antifeedant, attractant,bacteriocide, fungicide, hormone, insecticide, or rodenticide. Preferredantibiotics are cephalosporins and beta-lactams. Other preferredmetabolites are exo-metabolites. Examples of exo-metabolites areAurasperone B, Funalenone, Kotanin, Nigragillin, Orlandin, Othernaphtho-γ-pyrones, Pyranonigrin A, Tensidol B, Fumonisin B2 andOchratoxin A.

The biological compound may also be the product of a selectable marker.A selectable marker is a product of a polynucleotide of interest whichproduct provides for biocide or viral resistance, resistance to heavymetals, prototrophy to auxotrophs, and the like. Selectable markersinclude, but are not limited to, amdS (acetamidase), argB(ornithinecarbamoyltransferase), bar(phosphinothricinacetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),trpC (anthranilate synthase), ble (phleomycin resistance protein), hyg(hygromycin), NAT or NTC (Nourseothricin) as well as equivalentsthereof.

According to the invention, the compound of interest is preferably apolypeptide as described in the list of compounds of interest.

Preferably, the polypeptide is an enzyme as described in the list ofcompounds of interest. Preferably a glucose oxidase. In anotherembodiment the enzyme is a lipolytic enzyme.

According to another embodiment of the invention, the compound ofinterest is preferably a metabolite.

The mutant microbial cell may already be capable of producing thecompound of interest. The mutant microbial host cell may also beprovided with a homologous or heterologous nucleic acid construct thatencodes a polypeptide wherein the polypeptide may be the compound ofinterest or a polypeptide involved in the production of the compound ofinterest. The person skilled in the art knows how to modify a microbialhost cell such that it is capable of producing the compound of interest.

The term “nucleic acid construct” is herein referred to as a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which has been modified to containsegments of nucleic acid which are combined and juxtaposed in a mannerwhich would not otherwise exist in nature. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains all the control sequences required forexpression of a coding sequence, wherein said control sequences areoperably linked to said coding sequence.

The term “operably linked” is defined herein as a configuration in whicha control sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the production of an RNA or an mRNA and optionally of apolypeptide translated from said (m)RNA.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofmRNA and/or a polypeptide, either in vitro or in a host cell. Eachcontrol sequence may be native or foreign to the nucleic acid sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, Shine-Delgarno sequence, optimal translationinitiation sequences (as described in Kozak, 1991, J. Biol. Chem.266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, apre-pro-peptide sequence, a promoter, a signal sequence, and atranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Controlsequences may be optimized to their specific purpose. Preferredoptimized control sequences used in the present invention are thosedescribed in WO2006/077258, which is herein incorporated by reference.

The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

The control sequence may be an appropriate promoter sequence (promoter).

The control sequence may also be a suitable transcription terminator(terminator) sequence, a sequence recognized by a filamentous fungalcell to terminate transcription. The terminator sequence is operablylinked to the 3′-terminus of the nucleic acid sequence encoding thepolypeptide. Any terminator, which is functional in the cell, may beused in the present invention. The man skilled in the art knows whichtypes of terminators can be used in the microbial host cell as describedherein.

Preferred terminator sequences for filamentous fungal cells are obtainedfrom any terminator sequence of a filamentous fungal gene, morepreferably from Aspergillus genes, even more preferably from the gene A.oryzae TAKA amylase, the genes encoding A. niger glucoamylase (glaA), A.nidulans anthranilate synthase, A. niger alpha-glucosidase, trpC and/orFusarium oxysporum trypsin-like protease.

The control sequence may also be an optimal translation initiationsequences (as described in Kozak, 1991, J. Biol. Chem. 266:19867-19870),or a 5′-untranslated sequence, a non-translated region of a mRNA whichis important for translation by the mutated microbial host cell. Thetranslation initiation sequence or 5′-untranslated sequence is operablylinked to the 5′-terminus of the coding sequence encoding thepolypeptide. Each control sequence may be native or foreign to thenucleic acid sequence encoding the polypeptide. Control sequences may beoptimized to their specific purpose.

Suitable 5′-untranslated sequences may be those polynucleotidespreceeding the fungal amyloglucosidase (AG) gene, A. oryzae TAKA amylaseand Aspergillus triose phosphate isomerase genes and A. nigerglucoamylase glaA, alpha-amylase, xylanase and phytase encoding genes.

The control sequence may also be a non-translated region of a mRNA whichis important for translation by the mutated microbial host cell. Theleader sequence is operably linked to the 5′-terminus of the nucleicacid sequence encoding the polypeptide. Any leader sequence, which isfunctional in the cell, may be used in the present invention.

Leader sequences may be those originating from the fungalamyloglucosidase (AG) gene (glaA-both 18 and 24 amino acid versions e.g.from Aspergillus), the α-factor gene (yeasts e.g. Saccharomyces andKluyveromyces) or the α-amylase (amyE, amyQ and amyL) and alkalineprotease aprE and natural protease genes (Bacillus), or signal sequencesad described in WO2010/121933

Preferred leaders for filamentous fungal cells are obtained from thepolynucleotides preceding A. oryzae TAKA amylase and A. nidulans triosephosphate isomerase and A. niger glaA and phytase.

Other control sequences may be isolated from the Penicillium IPNS gene,or pcbC gene, the beta tubulin gene. All the control sequences cited inWO 01/21779 are herewith incorporated by reference.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′-terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the microbial host cell(mutated or parent) as a signal to add polyadenosine residues totranscribed mRNA. Any polyadenylation sequence, which is functional inthe cell, may be used in the present invention.

Preferred polyadenylation sequences for filamentous fungal cells areobtained from the polynucleotides encoding A. oryzae TAKA amylase, A.niger glucoamylase, A. nidulans anthranilate synthase, Fusariumoxysporum trypsin-like protease and A. niger alpha-glucosidase.

In a preferred embodiment, in the mutant microbial host cell accordingto the invention the at least one polynucleotide coding for the compoundof interest or the at least one polynucleotide coding for a compoundinvolved in the production of a compound of interest is operably linkedto a promoter, preferably to an inducible promoter.

The term “promoter” is defined herein as a DNA sequence that binds RNApolymerase and directs the polymerase to the correct downstreamtranscriptional start site of a nucleic acid sequence encoding abiological compound to initiate transcription. RNA polymeraseeffectively catalyzes the assembly of messenger RNA complementary to theappropriate DNA strand of a coding region. The term “promoter” will alsobe understood to include the 5′-non-coding region (between promoter andtranslation start) for translation after transcription into mRNA,cis-acting transcription control elements such as enhancers, and othernucleotide sequences capable of interacting with transcription factors.The promoter may be any appropriate promoter sequence suitable for aeukaryotic or prokaryotic host cell, which shows transcriptionalactivity, including mutant, truncated, and hybrid promoters, and may beobtained from polynucleotides encoding extra-cellular or intracellularpolypeptides either homologous (native) or heterologous (foreign) to thecell. The promoter may be a constitutive or inducible promoter.

Preferably the promoter is an inducible promoter. More preferably thepromoter is a carbohydrate inducible promoter. Carbohydrate induciblepromoters that are preferably used are selected from a starch-induciblepromoter (i.e. a promoter inducible by starch, a monomer, a dimer, aoligomer thereof, such as for example a maltose-inducible promoter, anisomaltose-inducible promoter), a cellulose-inducible promoter (i.e. apromoter inducible by cellulose, a monomer, a dimer and/or oligomerthereof, such as for example a cellobiose-inducible promoter, asophorose-inducible promoter), a hemicellulose inducible promoter (i.e.a promoter inducible by hemicellulose, a monomer, a dimer, and/or aoligomer thereof, such as e.g. a xylan-inducible promoter, anarabionose-inducible promoter, a xylose-inducible promoter), apectin-inducible promoter (i.e. a promoter inducible by pectin, amonomer, a dimer and/or an oligomer thereof such as for example agalacturonic acid-inducible promoter, a rhamnose-inducible promoter), anarabinan-inducible promoter (i.e. a promoter inducible by arabinan, amonomer, a dimer, and/or an oligomer thereof such as for example anarabinose-inducible promoter), a glucose-inducible promoter, alactose-inducible promoter, a galactose-inducible promoter. Otherinducible promoters are copper-, oleic acid-inducible promoters.

Promoters suitable in filamentous fungi are promoters which may beselected from the group, which includes but is not limited to promotersobtained from the polynucleotides encoding A. oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus gpdA promoter, A.niger neutral alpha-amylase, A. niger acid stable alpha-amylase, A.niger or A. awamori glucoamylase (glaA), A. niger or A. awamoriendoxylanase (xInA) or beta-xylosidase (xInD), T. reeseicellobiohydrolase I (CBHI), R. miehei lipase, A. oryzae alkalineprotease, A. oryzae triose phosphate isomerase, A. nidulans acetamidase,Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatumDania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusariumoxysporum trypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the polynucleotides encoding A.niger neutral alpha-amylase and A. oryzae triose phosphate isomerase),and mutant, truncated, and hybrid promoters thereof. Other examples ofpromoters are the promoters described in WO2006/092396 andWO2005/100573, which are herein incorporated by reference. An even otherexample of the use of promoters is described in WO2008/098933. Preferredcarbohydrate inducible promoters which can be used in filamentous fungiare the A. oryzae TAKA amylase, A. niger neutral alpha-amylase, A. nigeracid stable alpha-amylase, A. niger or A. awamori glucoamylase (glaA),A. niger or A. awamori endoxylanase (xInA) or beta-xylosidase (xInD),T., Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei cellobiohydrolase II,Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II,Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanaseIV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, aswell as the NA2-tpi promoter (a hybrid of the promoters from thepolynucleotides encoding A. niger neutral alpha-amylase and A. oryzaetriose phosphate isomerase) as defined above.

Examples of such promoters from Gram-positive microorganisms include,but are not limited to, gnt (gluconate operon promoter); penP fromBacillus licheniformis; glnA (glutamine synthetase); xylAB (xyloseoperon); araABD (L-arabinose operon) and Pspac promoter, a hybridSPO1/lac promoter that can be controlled by inducers such asisopropyl-β-D-thiogalactopyranoside [IPTG] ((Yansura D. G., Henner D. J.Proc Natl Acad Sci USA. 1984 81(2):439-443). Activators are alsosequence-specific DNA binding proteins that induce promoter activity.Examples of such promoters from Gram-positive microorganisms include,but are not limited to, two-component systems (PhoP-PhoR, DegU-DegS,Spo0A-Phosphorelay), LevR, Mry and GltC. (ii) Production of secondarysigma factors can be primarily responsible for the transcription fromspecific promoters. Examples from Gram-positive microorganisms include,but are not limited to, the promoters activated by sporulation specificsigma factors: σF, σE, σG and σK and general stress sigma factor, σB.The σB-mediated response is induced by energy limitation andenvironmental stresses (Hecker M, Völker U. Mol Microbiol. 1998;29(5):1129-1136.). (iii) Attenuation and antitermination also regulatestranscription. Examples from Gram-positive microorganisms include, butare not limited to, trp operon and sacB gene. (iv) Other regulatedpromoters in expression vectors are based the sacR regulatory systemconferring sucrose inducibility (Klier A F, Rapoport G. Annu RevMicrobiol. 1988; 42:65-95).

Suitable inducible promoters useful in bacteria, such as Bacilli,include: promoters from Gram-positive microorganisms such as, but arenot limited to, SP01-26, SP01-15, veg, pyc (pyruvate carboxylasepromoter), and amyE. Examples of promoters from Gram-negativemicroorganisms include, but are not limited to, tac, tet, trp-tet, lpp,lac, lpp-lac, laclq, T7, T5, T3, gal, trc, ara, SP6, λ-PR, and λ-PL.

Additional examples of promoters useful in bacterial cells, such asBacilli, include the α-amylase and SPo2 promoters as well as promotersfrom extracellular protease genes.

Other example of a suitable promoter are the promoter obtained from theE. coli lac operon. Another example is the promoter of the Streptomycescoelicolor agarase gene (dagA). Another example is the promoter of theBacillus lentus alkaline protease gene (aprH). Another example is thepromoter of the Bacillus licheniformis alkaline protease gene(subtilisin Carlsberg gene). Another example is the promoter of theBacillus subtilis levansucrase gene (sacB). Another example is thepromoter of the Bacillus subtilis alphaamylase gene (amyF). Anotherexample is the promoter of the Bacillus licheniformis alphaamylase gene(amyL). Another example is the promoter of the Bacillusstearothermophilus maltogenic amylase gene (amyM). Another example isthe promoter of the Bacillus amyloliquefaciens alpha-amylase gene(amyQ). Another example is a “consensus” promoter having the sequenceTTGACA for the “−35” region and TATAAT for the “−10” region. Anotherexample is the promoter of the Bacillus licheniformis penicillinase gene(penP). Another example are the promoters of the Bacillus subtilis xylAand xylB genes.

Preferably the promoter sequence is from a highly expressed gene.Examples of preferred highly expressed genes from which promoters may beselected and/or which are comprised in preferred predetermined targetloci for integration of expression constructs, include but are notlimited to genes encoding glycolytic enzymes such as triose-phosphateisomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH),phosphoglycerate kinases (PGK), pyruvate kinases (PYK or PKI), alcoholdehydrogenases (ADH), as well as genes encoding amylases, glucoamylases,proteases, xylanases, cellobiohydrolases, β-galactosidases, alcohol(methanol) oxidases, elongation factors and ribosomal proteins. Specificexamples of suitable highly expressed genes include e.g. the LAC4 genefrom Kluyveromyces sp., the methanol oxidase genes (AOX and MOX) fromHansenula and Pichia, respectively, the glucoamylase (glaA) genes fromA. niger and A. awamori, the A. oryzae TAKA-amylase gene, the A.nidulans gpdA gene and the T. reesei cellobiohydrolase genes.

Promoters which can be used in yeast include e.g. promoters fromglycolytic genes, such as the phosphofructokinase (PFK), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase(GPD, TDH3 or GAPDH), pyruvate kinase (PYK), phosphoglycerate kinase(PGK) promoters from yeasts or filamentous fungi; more details aboutsuch promoters from yeast may be found in (WO 93/03159). Other usefulpromoters are ribosomal protein encoding gene promoters, the lactasegene promoter (LAC4), alcohol dehydrogenase promoters (ADHI, ADH4, andthe like), and the enolase promoter (ENO). Other promoters, bothconstitutive and inducible, and enhancers or upstream activatingsequences will be known to those of skill in the art. The promoters usedin the host cells of the invention may be modified, if desired, toaffect their control characteristics. Suitable promoters in this contextinclude both constitutive and inducible natural promoters as well asengineered promoters, which are well known to the person skilled in theart. Suitable promoters in eukaryotic host cells may be GAL7, GAL10, orGAL1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO1,TPI1, and AOX1. Other suitable promoters include PDC1, GPD1, PGK1, TEF1,and TDH3. Examples of carbohydrate inducible promoters which can be usedare GAL promoters, such as GAL1 or GAL10 promoters.

All of the above-mentioned promoters are readily available in the art.

In a preferred embodiment, in the mutant microbial cell according to theinvention the at least one polynucleotide coding for a compound ofinterest or the at least one polynucleotide coding for a compoundinvolved in the production of a compound of interest is operably linkedto a carbohydrate inducible promoter, preferably a starch induciblepromoter, more preferably a promoter selected from a glucoamylasepromoter, acid stable amylase promoter, an alpha-amylase promoter andTAKA amylase promoter.

In order to facilitate expression, the polynucleotide encoding thepolypeptide being the compound of interest or the polypeptide involvedin the production of the compound of interest may be a syntheticpolynucleotide. The synthetic polynucleotides may be optimized in codonuse, preferably according to the methods described in WO2006/077258and/or PCT/EP2007/055943 (published as WO2008/000632), which are hereinincorporated by reference. PCT/EP2007/055943 addresses codon-pairoptimization. Codon-pair optimization is a method wherein the nucleotidesequences encoding a polypeptide have been modified with respect totheir codon-usage, in particular the codon-pairs that are used, toobtain improved expression of the nucleotide sequence encoding thepolypeptide and/or improved production of the encoded polypeptide. Codonpairs are defined as a set of two subsequent triplets (codons) in acoding sequence.

In order to facilitate expression and/or translation, the polynucleotideencoding the polypeptide being the compound of interest or encoding thepolypeptide involved in the production of the compound of interest maybe comprised in an expression vector such that the gene encoding thepolypeptide product is operably linked to the appropriate controlsequences for expression and/or translation in vitro, or in the mutantmicrobial host cell.

The expression vector may be any vector (e.g., a plasmid or virus),which can be conveniently subjected to recombinant DNA procedures andcan bring about the expression of the polynucleotide encoding thepolypeptide. The choice of the vector will typically depend on thecompatibility of the vector with the cell into which the vector is to beintroduced. The vectors may be linear or closed circular plasmids. Thevector may be an autonomously replicating vector, i.e., a vector, whichexists as an extra-chromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. An autonomously maintained cloning vector may comprise theAMA1-sequence (see e.g. Aleksenko and Clutterbuck (1997), Fungal Genet.Biol. 21: 373-397).

Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. The integrativecloning vector may integrate at random or at a predetermined targetlocus in the chromosomes of the host cell. In a preferred embodiment ofthe invention, the integrative cloning vector comprises a DNA fragment,which is homologous to a DNA sequence in a predetermined target locus inthe genome of host cell for targeting the integration of the cloningvector to this predetermined locus. In order to promote targetedintegration, the cloning vector is preferably linearized prior totransformation of the cell. Linearization is preferably performed suchthat at least one but preferably either end of the cloning vector isflanked by sequences homologous to the target locus. The length of thehomologous sequences flanking the target locus is preferably at least 30bp, preferably at least 50 bp, preferably at least 0.1 kb, evenpreferably at least 0.2 kb, more preferably at least 0.5 kb, even morepreferably at least 1 kb, most preferably at least 2 kb. Preferably, theefficiency of targeted integration into the genome of the host cell,i.e. integration in a predetermined target locus, is increased byaugmented homologous recombination abilities of the host cell.

Preferably, the homologous flanking DNA sequences in the cloning vector,which are homologous to the target locus, are derived from a highlyexpressed locus meaning that they are derived from a gene, which iscapable of high expression level in the host cell. A gene capable ofhigh expression level, i.e. a highly expressed gene, is herein definedas a gene whose mRNA can make up at least 0.5% (w/w) of the totalcellular mRNA, e.g. under induced conditions, or alternatively, a genewhose gene product can make up at least 1% (w/w) of the total cellularprotein, or, in case of a secreted gene product, can be secreted to alevel of at least 0.1 g/l (as described in EP 357 127 B1).

A number of preferred highly expressed fungal genes are given by way ofexample: the amylase, glucoamylase, alcohol dehydrogenase, xylanase,glyceraldehyde-phosphate dehydrogenase or cellobiohydrolase (cbh) genesfrom Aspergilli, Chrysosporium or Trichoderma. Most preferred highlyexpressed genes for these purposes are a glucoamylase gene, preferablyan A. niger glucoamylase gene, an A. oryzae TAKA-amylase gene, an A.nidulans gpdA gene, a Trichoderma reesei cbh gene, preferably cbh1, aChrysosporium lucknowensecbh gene or a cbh gene from P. chrysogenum.

More than one copy of a nucleic acid sequence may be inserted into themutated microbial host cell to increase production of the product(over-expression) encoded by said sequence. This can be done, preferablyby integrating into its genome copies of the DNA sequence, morepreferably by targeting the integration of the DNA sequence at one ofthe highly expressed loci defined in the former paragraph.Alternatively, this can be done by including an amplifiable selectablemarker gene with the nucleic acid sequence where cells containingamplified copies of the selectable marker gene, and thereby additionalcopies of the nucleic acid sequence, can be selected for by cultivatingthe cells in the presence of the appropriate selectable agent. Toincrease even more the number of copies of the DNA sequence to be overexpressed the technique of gene conversion as described in WO98/46772may be used.

The vector system may be a single vector or plasmid or two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon.

The vectors preferably contain one or more selectable markers, whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like. Theselectable marker may be introduced into the cell on the expressionvector as the expression cassette or may be introduced on a separateexpression vector.

A selectable marker for use in a filamentous fungal cell may be selectedfrom the group including, but not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar(phosphinothricinacetyltransferase), bleA (phleomycin binding), hygB(hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),NAT or NTC (Nourseothricin) and trpC (anthranilate synthase), as well asequivalents from other species. Preferred for use in an Aspergillus andPenicillium cell are the amdS (see for example EP 635574 B1,EP0758020A2, EP1799821A2, WO 97/06261A2) and pyrG genes of A. nidulansor A. oryzae and the bar gene of Streptomyces hygroscopicus. Morepreferably an amdS gene is used, even more preferably an amdS gene fromA. nidulans or A. niger. A most preferred selectable marker gene is theA. nidulans amdS coding sequence fused to the A. nidulans gpdA promoter(see EP 635574 B1). Other preferred AmdS markers are those described inWO2006/040358. AmdS genes from other filamentous fungi may also be used(WO 97/06261).

Markers which can be used in bacteria include ATP synthetase, subunit 9(oliC), orotidine-5′-phosphatedecarboxylase (pvrA), the bacterial G418resistance gene (this may also be used in yeast, but not in filamentousfungi), the ampicillin resistance gene (E. coli), resistance genes for,neomycin, kanamycin, tetracycline, spectinomycin, erythromycin,chloramphenicol, phleomycin (Bacillus) and the E. coli uidA gene, codingfor β-glucuronidase (GUS). Vectors may be used in vitro, for example forthe production of RNA or used to transfect or transform a host cell.

Versatile marker genes that can be used for transformation of mostfilamentous fungi and yeasts such as acetamidase genes or cDNAs (theamdS, niaD, facA genes or cDNAs from A. nidulans, A. oryzae or A.niger), or genes providing resistance to antibiotics like G418,hygromycin, bleomycin, kanamycin, methotrexate, phleomycin orbenomylresistance (benA). Alternatively, specific selection markers can be usedsuch as auxotrophic markers which require corresponding mutant hoststrains: e.g. D-alanine racemase (from Bacillus), URA3 (from S.cerevisiae or analogous genes from other yeasts), pyrG or pyrA (from A.nidulans or A. niger), argB (from A. nidulans or A. niger) or trpC. In apreferred embodiment the selection marker is deleted from thetransformed host cell after introduction of the expression construct soas to obtain transformed host cells capable of producing the polypeptidewhich are free of selection marker genes.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g. Sambrook & Russell, MolecularCloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor,N.Y., 2001; and Ausubel et al., Current Protocols in Molecular Biology,Wiley InterScience, NY, 1995).

Furthermore, standard molecular cloning techniques such as DNAisolation, gel electrophoresis, enzymatic restriction modifications ofnucleic acids, Southern analyses, transformation of cells, etc., areknown to the skilled person and are for example described by Sambrook etal. (1989) “Molecular Cloning: a laboratory manual”, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y. and Innis et al. (1990) “PCRprotocols, a guide to methods and applications” Academic Press, SanDiego.

A nucleic acid may be amplified using cDNA, mRNA or alternatively,genomic DNA, as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis.

Preferably, the mutant microbial host cell is modified to improve theexpression of the polynucleotides to enhance production of thepolypeptides being the compound of interest or a polypeptide involved inthe production of a compound of interest.

Preferably, the efficiency of targeted integration into the genome ofthe host cell, i.e. integration in a predetermined target locus, isincreased by augmented homologous recombination abilities of the hostcell. Such phenotype of the cell preferably involves a deficient hdfA orhdfB as described in WO2005/095624. WO2005/095624 discloses a preferredmethod to obtain a filamentous fungal cell comprising increasedefficiency of targeted integration.

Optionally, the host cell has been modified to comprise an elevatedunfolded protein response (UPR) to enhance production abilities of apolypeptide of interest. UPR may be increased by techniques described inUS2004/0186070A1 and/or US2001/0034045A1 and/or WO01/72783A2 and/orWO2005/123763. More specifically, the protein level of HAC1 and/or IRE1and/or PTC2 may be modulated, and/or the SEC61 protein may be engineeredin order to obtain a host cell having an elevated UPR.

The person skilled in the art knows how to transform cells with the oneor more expression cassettes and the selectable marker. For example, theskilled person may use one or more expression vectors, wherein the oneor more cloning vectors comprise the expression cassettes and theselectable marker.

Transformation of the mutant microbial host cell may be conducted by anysuitable known methods, including e.g. electroporation methods, particlebombardment or microprojectile bombardment, protoplast methods andAgrobacterium mediated transformation (AMT). Preferably the protoplastmethod is used. Procedures for transformation are described by J. R. S.Fincham, Transformation in fungi. 1989, Microbiological reviews. 53,148-170.

Transformation of the mutant microbial host cell by introduction of apolynucleotide an expression vector or a nucleic acid construct into thecell is preferably performed by techniques well known in the art (seeSambrook & Russell; Ausubel, supra). Transformation may involve aprocess consisting of protoplast formation, transformation of theprotoplasts, and regeneration of the cell wall in a manner known per se.Suitable procedures for transformation of Aspergillus cells aredescribed in EP 238 023 and Yelton et al., 1984, Proceedings of theNational Academy of Sciences USA 81:1470-1474. Suitable procedures fortransformation of Aspergillus and other filamentous fungal host cellsusing Agrobacterium tumefaciens are described in e.g. De Groot et al.,Agrobacterium tumefaciens-mediated transformation of filamentous fungi.Nat Biotechnol. 1998, 16:839-842. Erratum in: Nat Biotechnol 199816:1074. A suitable method of transforming Fusarium species is describedby Malardier et al., 1989, Gene 78:147156 or in WO 96/00787. Othermethods can be applied such as a method using biolistic transformationas described in: Christiansen et al., Biolistic transformation of theobligate plant pathogenic fungus, Erysiphe graminis f.sp. hordei. 1995,Curr Genet. 29:100-102. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

In order to enhance the amount of copies of the polynucleotide codingfor the compound of interest or coding for a compound involved in theproduction by the cell of the compound of interest (the gene) in themutated microbial host cell, multiple transformations of the host cellmay be required. In this way, the ratios of the different enzymesproduced by the host cell may be influenced. Also, an expression vectormay comprise multiple expression cassettes to increase the amount ofcopies of the polynucleotide(s) to be transformed.

Another way could be to choose different control sequences for thedifferent polynucleotides, which—depending on the choice—may cause ahigher or a lower production of the desired polypeptide(s).

The cells transformed with the selectable marker can be selected basedon the presence of the selectable marker. In case of transformation of(Aspergillus) cells, usually when the cell is transformed with allnucleic acid material at the same time, when the selectable marker ispresent also the polynucleotide(s) encoding the desired polypeptide(s)are present.

The invention also provides a method of producing a mutant microbialhost cell according to the invention comprising the steps of:

-   -   a. providing a parent microbial host cell as described herein;    -   b. modifying the parent microbial host cell, preferably        modifying the genome of the parent microbial host cell, to yield        a mutant microbial host cell as described herein which is        deficient in the production of a polypeptide as described herein        selected from the group consisting of:        -   (i) a polypeptide according to SEQ ID NO: 3 or a polypeptide            at least 70% identical thereto, preferably a polypeptide at            least 70% identical thereto having at least one activity of            the polypeptide according to SEQ ID NO:3;        -   (ii) a mature polypeptide comprised in SEQ ID NO: 3 or a            polypeptide at least 70% identical thereto, preferably a            polypeptide at least 70% identical thereto and having at            least one activity of the mature polypeptide comprised in            SEQ ID NO:3;        -   (iii) a polypeptide encoded by a polynucleotide according to            SEQ ID NO: 1 or 2 or encoded by a polynucleotide at least            70% identical to SEQ ID NO: 1 or 2, wherein said polypeptide            encoded by a polynucleotide according to SEQ ID NO: 1 or 2            has preferably at least one activity of the polypeptide            encoded by the polynucleotide according to SEQ ID NO: 1 or            2;        -   (iv) a polypeptide encoded by a polynucleotide capable of            hybridising a polynucleotide according to SEQ ID NO: 1 or 2            or capable of hybridising to the complementary strand of SEQ            ID NO: 1 or 2, wherein said polypeptide has preferably at            least one activity of the polypeptide encoded by the            polynucleotide according to SEQ ID NO: 1 or 2;        -   if compared with the parent microbial host cell and measured            under the same conditions.

Within this context it will be clear to those skilled in the art thatthe specific embodiments applicable to the mutant microbial host cellaccording to the invention may also be applicable to the other aspectsof the invention.

The invention further provides a method for the production of a compoundof interest by microbial fermentation comprising:

-   -   a. providing a mutant microbial host cell according to the        invention capable of expressing the compound of interest,    -   b. culturing said microbial host cell under conditions conducive        to the expression of the compound of interest,    -   c. optionally isolating the compound of interest from the        culture medium.

In step a. a mutant microbial host cell can be a mutant host cell asdescribed herein.

In step b. the mutant microbial host cell of step a. is cultured underconditions conducive to the expression of the compound of interest asdescribed herein. The mutant microbial cells are cultivated in anutrient medium suitable for production of the compound of interestusing methods known in the art. For example, the cells may be cultivatedby shake flask cultivation, small-scale or large-scale fermentation(including continuous, batch, fed-batch, or solid state fermentations)in laboratory or industrial fermentors performed in a suitable mediumand under conditions allowing the compound of interest to be producedand/or isolated. The cultivation takes place in a suitable nutrientmedium comprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art (see, e.g., Bennett, J. W. and LaSure, L.,eds., More Gene Manipulations in Fungi, Academic Press, CA, 1991).Suitable media are available from commercial suppliers or may beprepared using published compositions (e.g., in catalogues of theAmerican Type Culture Collection). If the compound of interest issecreted into the nutrient medium, the compound can be isolated directlyfrom the medium. If the compound of interest is not secreted, it can beisolated from cell lysates.

In step c. the compound of interest may be optionally isolated. Thecompound of interest as described herein may be isolated by methodsknown in the art. For example, the compound of interest may be isolatedfrom the nutrient medium by conventional procedures including, but notlimited to, centrifugation, filtration, extraction, spray drying,evaporation, or precipitation. The isolated compound of interest maythen be further purified by a variety of procedures known in the artincluding, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation), orextraction (see, e.g., Protein Purification, J.-C. Janson and LarsRyden, editors, VCH Publishers, New York, 1989). In some applicationsthe compound of interest may be used without substantial isolation fromthe culture broth; separation of the culture medium from the biomass maybe adequate.

In a preferred embodiment of the method for the production of a compoundof interest according to the invention, the yield of the compound ofinterest is increased if compared to the yield of a method forproduction of a compound of interest where a parent microbial host cellwhich has not been modified is used, measured under the same conditions.Preferably, it increases with at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least9%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90% or at least100%. More preferably, with at least 110%, at least 120%, at least 130%,at least 140%, at least 150%, at least 160%, at least 170%, at least180%, at least 190% or at least 200%. Even more preferably with at least210%, at least 220%, at least 230%, at least 240%, at least 250%, atleast 260%, at least 270%, at least 280%, at least 290% or at least300%.

A mutant microbial host cell as defined herein may be used in the methodfor the production of a compound of interest as described herein.

The compound of interest produced in the method for the production of acompound of interest by microbial fermentation may be any compound ofinterest as described herein.

Preferred Embodiments of the Invention

1. A mutant microbial host cell which has been modified, preferably inits genome, to result in a deficiency in the production of a polypeptideselected from the group consisting of:

-   -   a. a polypeptide according to SEQ ID NO: 3 or a polypeptide at        least 70% identical thereto and preferably having at least one        activity of the polypeptide according to SEQ ID NO:3;    -   b. a mature polypeptide comprised in SEQ ID NO: 3 or a        polypeptide at least 70% identical thereto and preferably having        at least one activity of the mature polypeptide comprised in SEQ        ID NO:3;    -   c. a polypeptide encoded by a polynucleotide according to SEQ ID        NO: 1 or 2 or encoded by a polynucleotide at least 70% identical        to SEQ ID NO: 1 or 2, wherein said polypeptide encoded by a        polynucleotide according to SEQ ID NO: 1 or 2 has preferably at        least one activity of the polypeptide encoded by the        polynucleotide according to SEQ ID NO: 1 or 2;    -   d. a polypeptide encoded by a polynucleotide capable of        hybridising to a polynucleotide according to SEQ ID NO: 1 or 2        or capable of hybridising to the complementary strand of SEQ ID        NO: 1 or 2, wherein said polypeptide has preferably at least one        activity of the polypeptide encoded by the polynucleotide        according to SEQ ID NO: 1 or 2;    -   if compared with a parent microbial host cell which has not been        modified and measured under the same conditions.

2. A mutant microbial host cell according to embodiment 1 wherein themature polypeptide comprised in SEQ ID NO: 3 is the mature polypeptideaccording to SEQ ID NO: 4.

3. A mutant microbial host cell according to any one of embodiment 1 or2 wherein the polypeptide according to embodiment 1 a. to 1.d has anenzymatic activity which is a glycoside hydrolase activity, morepreferably an enzymatic activity selected from the group consisting of:α-amylase activity [EC 3.2.1.1], isoamylase activity, inulinaseactivity, invertase activity [EC 3.2.1.26], maltase activity [EC3.2.1.20], isomaltase activity, pullulanase activity, glucoamylaseactivity, cyclodextrinase activity, chitosanase activity, dextranaseactivity, sucrase-isomaltase activity, α-glucosidase activity, glycogendebranching enzymatic activity.

4. A mutant microbial host cell according to any one of embodiments 1 to2 wherein the polypeptide according to embodiment 1 a. to 1.d has anenzymatic activity which is α-gluconotransferase activity, saidenzymatic activity is preferably a glycoside transferase or glycosidesynthase activity, more preferably an enzymatic activity selected fromthe group consisting of: glycogen branching enzymatic activity,α-1,3-glucan synthase enzymatic activity [EC 2.4.1.183], α-1,4-glucansynthase activity, α-1,6-glucan synthase activity, β-1,3-glucan synthaseactivity, β-1,4-glucan synthase activity, β-1,6-glucan synthaseactivity, glucoamylase activity, maltopentaose-forming amylase activity,maltohexaose-forming amylase activity, α-glucosidase activity,α-glucosidase II activity, α-xylosidase activity.

5. The mutant microbial host cell according to any one of embodiments 1to 5 wherein the modification comprises:

-   -   a) a modification which results in a reduced or no production of        a polypeptide as defined in embodiment 1 a. to 1 d. if compared        to the parent microbial host cell that has not been modified,        when analysed under the same conditions and/or    -   b) a modification which results in a polypeptide derived from        the polypeptide as defined in embodiment 1 a. to 1.d with        decreased or no activity if compared to the parent microbial        host cell that has not been modified, when analysed under the        same conditions.

6. The mutant microbial host cell according to any one of embodiments 1to 5 wherein the mutant microbial host cell

-   -   a. produces less polypeptide as defined in embodiment 1 a. to        1 d. or it produces no polypeptide as defined in embodiment 1 a.        to 1 d if compared with the parent microbial host cell which has        not been modified and measured under the same conditions; and/or    -   b. produces a polypeptide derived from the polypeptide as        defined in embodiment 1 a. to 1 d with decreased or no activity        if compared to the parent microbial host cell that has not been        modified, when analysed under the same conditions.

7. The mutant microbial host cell according to any one of embodiments 1to 6 wherein the mutant microbial host cell produces 1% less polypeptideas defined in embodiment 1 a. to 1 d. if compared with the parentmicrobial host cell which has not been modified and measured under thesame conditions, at least 5% less, at least 10% less, at least 20% less,at least 30% less, at least 40% less, at least 50% less, at least 60%less, at least 70% less, at least 80% less, at least 90% less, at least91% less, at least 92% less, at least 93% less, at least 94% less atleast 95% less, at least 96% less, at least 97% less, at least 98% less,at least 99% less, or at least 99.9% less, preferably the mutantmicrobial host cell produces substantially no polypeptide as defined inclaim 1 a. to 1 d. if compared with the parent microbial host cell whichhas not been modified and measured under the same conditions.

8. The mutant microbial host cell according to any one of embodiments 1to 7 wherein the mutant microbial host cell produces a polypeptidederived from the polypeptide as defined in embodiment 1 a. to 1 d. with1% less (enzymatic) activity, if compared with the parent microbial hostcell which has not been modified and measured under the same conditions,at least 5% less activity, at least 10% less activity, at least 20% lessactivity, at least 30% less activity, at least 40% less activity, atleast 50% less activity, at least 60% less activity, at least 70% lessactivity, at least 80% less activity, at least 90% less activity, atleast 91% less activity, at least 92% less activity, at least 93% lessactivity, at least 94% less activity, at least 95% less activity, atleast 96% less activity, at least 97% less activity, at least 98% lessactivity, at least 99% less activity, or at least 99.9% less activity,preferably the mutant microbial host cell produces a polypeptide derivedfrom a polypeptide as defined in claim 1 a. to 1 d. with substantiallyno activity if compared with the parent microbial host cell which hasnot been modified and analysed under the same conditions.

9. The mutant microbial host cell according to any one of embodiments 1to 8 wherein the modification in its genome is selected from:

-   -   a) a full or partial deletion of a polynucleotide as defined in        embodiment 1 c. or 1 d.;    -   b) a full or partial replacement of a polynucleotide as defined        in embodiment 1 c. or 1 d. with a polynucleotide sequence which        does not code for a polypeptide as defined in embodiment 1 a. to        1 d. or which code for a partially or fully inactive form of a        polypeptide as defined in embodiment 1 a. to 1 d.;    -   c) a disruption of a polynucleotide as defined in embodiment        1 c. or 1 d. by the insertion of one or more nucleotides in the        polynucleotide sequence and consequent partial or full        inactivation of a polypeptide as defined in embodiment 1 a. to 1        d.

10. The mutant microbial host cell according to any one of embodiments 1to 9 wherein the modification which results in a reduced or noproduction of a polypeptide as defined in embodiment 1 a. to 1 d. is dueto a reduced production of the mRNA encoding said polypeptide.

11. The mutant microbial host cell according to any one of embodiments 1to 10 comprising at least one polynucleotide coding for a compound ofinterest or at least one polynucleotide coding for a compound involvedin the production of a compound of interest.

12. The mutant microbial host cell according to embodiment 11 whereinthe at least one polynucleotide coding for the compound of interest orthe at least one polynucleotide coding for a compound involved in theproduction of a compound of interest is operably linked to a promoter,preferably to an inducible promoter.

13. The mutant microbial host cell according to any one of embodiments 1to 12 wherein the promoter is a carbohydrate inducible promoter,preferably a promoter selected from a starch inducible promoter, morepreferably a glucoamylase promoter, acid stable amylase promoter, analpha-amylase promoter and TAKA amylase promoter.

14. The mutant microbial host cell according to any one of embodiments 1to 13 which is a eukaryotic cell, more preferably a fungal cell, evenmore preferably the mutant microbial host cell is a filamentous fungus.

15. The mutant microbial host cell according to embodiment 14 which is afilamentous fungus selected from Aspergillus, Acremonium,Myceliophthora, Thielavia Chrysosporium, Penicillium, Talaromyces,Rasamsonia, Fusarium or Trichoderma, preferably a species of Aspergillusniger, Aspergillus awamori, Aspergillus foetidus, Aspergillus sojae,Aspergillus fumigatus, Aspergillus oryzae, Acremonium alabamense,Myceliophthora thermophila, Thielavia terrestris, Chrysosporiumlucknowense, Fusarium oxysporum, Rasamsonia emersonfi, Talaromycesemersonii, Trichoderma reesei or Penicillium chrysogenum.

16. A method of producing a mutant microbial host cell comprising thesteps of:

-   -   a. providing a parent microbial host cell;    -   b. modifying the parent microbial host cell, preferably        modifying the genome of the parent microbial host cell, to yield        a mutant microbial host cell which is deficient in the        production of a polypeptide selected from the group consisting        of:        -   (i) a polypeptide according to SEQ ID NO: 3 or a polypeptide            at least 70% identical thereto, preferably a polypeptide at            least 70% identical thereto having at least one activity of            the polypeptide according to SEQ ID NO:3;        -   (ii) a mature polypeptide comprised in SEQ ID NO: 3 or a            polypeptide at least 70% identical thereto, preferably a            polypeptide at least 70% identical thereto and having at            least one activity of the mature polypeptide comprised in            SEQ ID NO:3;        -   (iii) a polypeptide encoded by a polynucleotide according to            SEQ ID NO: 1 or 2 or encoded by a polynucleotide at least            70% identical to SEQ ID NO: 1 or 2, wherein said polypeptide            encoded by a polynucleotide according to SEQ ID NO: 1 or 1            has preferably at least one activity of the polypeptide            encoded by the polynucleotide according to SEQ ID NO: 1 or            2;        -   (iv) a polypeptide encoded by a polynucleotide capable of            hybridising a polynucleotide according to SEQ ID NO: 1 or 2            or capable of hybridising to the complementary strand of SEQ            ID NO: 1 or 2, wherein said polypeptide has preferably at            least one activity of the polypeptide encoded by the            polynucleotide according to SEQ ID NO: 1 or 2;        -   if compared with the parent microbial host cell and measured            under the same conditions.

17. The method according to embodiment 16 wherein the mutant microbialhost cell is a mutant microbial host cell according to any one ofembodiments 1 to 15.

18. A method for the production of a compound of interest by microbialfermentation comprising:

-   -   a. providing a mutant microbial host cell according to any one        of embodiments 1 to 15 or produced by a method according to        embodiments 16 or 17 capable of expressing the compound of        interest,    -   b. culturing said mutant microbial host cell under conditions        conducive to the expression of the compound of interest,    -   c. optionally isolating the compound of interest from the        culture medium.

19. The method according to embodiment 18 wherein the compound ofinterest is a biological compound selected from the group consisting ofbiomass, a biopolymer, a metabolite, preferably the compound of interestis selected from a biopolymer or a metabolite.

20. The method according to embodiment 19 wherein the biopolymer isselected from a nucleic acid, a polyamine, a polyol, a polypeptide (suchas a protein, preferably an enzyme) or a polyamide, or a polysaccharideor a metabolite is selected from a primary or secondary metabolite.

21. The method according to embodiment 20 wherein the compound ofinterest is an enzyme, preferably glucose oxidase.

22. The method according to any one of embodiments 18 to 21 wherein theyield of the compound of interest is increased if compared to the yieldof a method for production of a compound of interest where a parentmicrobial host cell which has not been modified is used, measured underthe same conditions, preferably wherein the yield increases with atleast 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90% or at least 100%, more preferably, with at least110%, at least 120%, at least 130%, at least 140%, at least 150%, atleast 160%, at least 170%, at least 180%, at least 190% or at least200%, even more preferably with at least 210%, at least 220%, at least230%, at least 240%, at least 250%, at least 260%, at least 270%, atleast 280%, at least 290% or at least 300%.

Hereafter the invention will be illustrated by examples which howevershould not be interpreted as limiting the scope of the invention.

Strains

WT 1: This Aspergillus niger strain is used as a wild-type strain. Thisstrain is deposited at the CBS Institute under the deposit number CBS513.88.

GBA 306: The construction of GBA 306 using WT1 as starting strain hasbeen described in detail in WO2011/009700. This GBA 306 strain has thefollowing genotype: ΔglaA, ΔpepA, ΔhdfA, an adapted BamHI amplicon,ΔamyBII, ΔamyBI, and ΔamyA.

PGOX-2: This A. niger strain is a GBA306 strain expressing thePenicillium chrysogenum glucose oxidase enzyme. The PGOX-2 strain wasconstructed using the pGBTOPGOX-3 expression vector (see FIG. 1—pGBTOP12expression vector (WO2011/009700) with a codon pair optimizedPenicillium chrysogenum glucose oxidase (as depicted in SEQ ID NO: 29and with a protein sequence as depicted in SEQ ID NO: 30 ofWO2012/001169) coding sequence cloned in), which was introduced byco-transformation with the amdS selectable marker-gene containing vectorpGBAAS-3 using the method as described in WO2011/009700 andWO2012/001169. After transformation and counter-selection (as alsodescribed in WO98/46772 and WO99/32617), followed by selection ofstrains with multiple copies, 1 multi-copy enzyme-producing strain wasselected and named PGOX-2. This strain is used as the glucose oxidaseenzyme producing strain in subsequent experiments.

PLA-2 and LIP-2: The porcine phospholipase A2 (PLA2) protein and alipolytic L01 enzyme (having amino acid sequence according to SEQ ID NO:2 and coded by the polynucleotide sequence of SEQ ID NO: 1 as describedin WO2009/106575) were selected as model proteins for enzyme expressionin the A. niger GBA 306. Both enzymes were also expressed in a differentA. niger background, but expression cassettes and coding sequences wereessentially similar as described in Example 1 of WO2012001169.

Porcine phospholipase A2 (PLA2) protein (Roberts I. N., Jeenes D. J.,MacKenzie D. A., Wilkinson A. P., Sumner I. G. and Archer D. B. (1992)“Heterologous gene expression in Aspergillus niger: aglucoamylase-porcine pancreatic phospholipase A2 fusion protein issecreted and processed to yield mature enzyme” Gene 122: 155-161) wasselected as a model protein for enzyme expression in the A. nigerstrains. The fragment for overexpression of PLA2 was made as a fusion ofproPLA2 with a native glucoamylase A gene of A. niger and was preparedin principle as described by Roberts et al. (1992) and WO2012001169. Thekex2 cleavage site (KR) between GLA and porPLA2 is removed, so that theGLA-proPLA2 fusion protein encoded is as set out in SEQ ID NO: 20.

This glaA-pla2 fusion gene encoding the above mentioned protein iscloned into an A. niger pGBTOP-12 expression vector using the sametechniques as described in WO 98/46772 and WO 99/32617, generatingpGBTOPPLA-2 (FIG. 3). The gene encoding the lipolytic enzyme L01 wascloned into an A. niger pGBTOP-12 expression vector using the techniquesas described in WO 98/46772 and WO 99/32617, under the control of theglucoamylase promoter essentially as described in WO2012001169, yieldingpGBTOPLIP-2 (FIG. 2).

Enzyme producing strains for the lipolytic enzyme and theglucoamylase-porcine pancreatic phospholipase A2 fusion protein wereconstructed by co-transformation of the GBA 306 strain with the amdSselectable marker-gene containing vector pGBAAS-3 and the pGBTOPLIP-2and pGBTOPPLA-2 vector, respectively and subsequent selection oftransformants. The transformation and counterselection procedure (asdescribed in WO98/46772 and WO99/32617), followed by selection ofstrains resulted in (multicopy) strains producing lipase andglucoamylase-porcine pancreatic phospholipase A2 fusion proteinproducing strains. For each strain background, 1 high-copyenzyme-producing strain for the GBA 306 background was selected andnamed LIP-2 and PLA-2. These strains were used as the respective enzymeproducing strains in subsequent experiments.

Molecular Biology Techniques

In these strains, using molecular biology techniques known to theskilled person (see: Sambrook & Russell, Molecular Cloning: A LaboratoryManual, 3rd Ed., CSHL Press, Cold Spring Harbor, N.Y., 2001), severalgenes were over expressed and others were down regulated as describedbelow. Examples of the general design of expression vectors for geneover expression and disruption vectors for down-regulation,transformation, use of markers and selective media can be found inWO199846772, WO199932617, WO2001121779, WO2005095624, WO2006040312, EP635574B, WO2005100573, WO2011009700 and WO2012001169. All genereplacement vectors comprise approximately 1-2 kb flanking regions ofthe respective ORF sequences, to target for homologous recombination atthe predestined genomic loci. In addition, they contain the A. nidulansbi-directional amdS selection marker for transformation, in-betweendirect repeats. The method applied for gene deletion in all examplesherein uses linear DNA, which integrates into the genome at thehomologous locus of the flanking sequences by a double cross-over, thussubstituting the gene to be deleted by the amdS gene. Aftertransformation, the direct repeats allow for the removal of theselection marker by a (second) homologous recombination event. Theremoval of the amdS marker can be done by plating on fluoro-acetamidemedia, resulting in the selection of marker-gene-free strains. Usingthis strategy of transformation and subsequent counter-selection, whichis also described as the “MARKER-GENE FREE” approach in EP 0 635 574,the amdS marker can be used indefinitely in strain modificationprograms.

A. niger Shake Flask Fermentations

A. niger strains were pre-cultured and cultured at 34° C. and 170 rpm asdescribed in WO2010/102982. Pre-culture was in 20 ml CSL pre-culturemedium and after overnight growth 10 ml of this culture was transferredto 100 ml fermentation medium (FM) as described in more detail inWO2010/102982 with a cultivation time as indicated in the examples.

A. niger 24-Well Microtiterplate Fermentations

24-well microtiterplates containing 4 ml FM per well were inoculatedwith 1×10⁵-5×10⁵ A. niger spores. The plates were incubated at 34° C.,550 rpm and 80% humidity for 120 hours.

Enzyme Activity Measurements

Glucose oxidase (GOX) activity and the GOX activity plate assay (usingo-anisidine) were measured as described in Witteveen et al. 1990,“Glucose oxidase overproducing and negative mutants of Aspergillusniger”, Appl Microbiol Biotechnol 33:683-686.

To determine phospholipase PLA2 activity (PLA2) in Aspergillus nigerculture broth spectrophotometrically, an artificial substrate is used:1,2-dithiodioctanoyl phophatidylcholine (diC8, substrate). More detailsof this assay are described in WO2006/040312.

Samples can contain the (partially) inactive (non-processed) form ofPLA2=pro-PLA2. Trypsin is applied to clip off the pro-sequence fromphospholipase A2. Treatment of pro-PLA2 with trypsin results in acomplete activation of PLA2 present in the supernatants. Enzymaticactivity after trypsin treatment is expressed in CPU (ChromogenicPhospholipase A2 Unit) or PLA2 activity (relative CPU activity)

Lipase activity can be measured as described under “activitymeasurements” section of WO2009/106575.

Example 1 Construction Approach of Aspergillus niger PGOX-2 Strains,Containing Glycoside Hydrolase Gene Deletions

To be able to disrupt the glycoside hydrolase (GH)-related genes (alsoknown under the gene codes: An01g10930, An04g06920, and An09g03070encoding putative α-glucan synthase and/or (putative) α-glucosidaseenzymes of the GH31 or GH13 family and possibly involved in starchdegradation or cell wall alpha-glucan synthesis), a gene replacementvector was designed for each of the three genes as described above.Details of the amylase encoding genes can be found in Table 1.

TABLE 1 Gene and strain details for respective GH disruption strainconstructed Strain code for GH gene Disruption PCR results disruptionstrain disrupted vector amdS GH gene PGOX-2 − − − − PGOX-2_AMY1AgdB-An01g10930 pGBDEL-AMY1 + − PGOX-2_AMY2 AgdA-An04g06920pGBDEL-AMY2 + − PGOX-2_AMY4 AgsE-An09g03070 pGBDEL-AMY4 + − LIP-2 − − −− LIP-2_AMY4 AgsE-An09g03070 pGBDEL-AMY4 + − PLA-2 − − − − PLA-2_AMY4AgsE-An09g03070 pGBDEL-AMY4 + −

Vector pGBDEL-AMY1 (FIG. 4) and the other pGBDEL variants, whichcomprise approximately 1 kb flanking regions of the respective amylaseencoding ORF's for homologous recombination, were used to transformAspergillus niger PGOX-2. After verification of the truthfulrecombination events and correctness of the strains, the resultingcorrect strains PGOX-2, PGOX-2_AMY1, PGOX-2_AMY2, PGOX-2_AMY4-1 andPGOX-2_AMY4-2 were selected as representative strains with therespective GH genes (Table 1) inactivated in the PGOX-2 strainbackground.

The same approach was followed for Aspergillus niger LIP-2 and PLA-2strains. This resulted in the strains LIP-2, LIP-2_AMY4-1, LIP-2_AMY4-2,PLA-2 and PLA-2_AMY4 with the respective GH genes (Table 1) inactivatedin the LIP-2 and PLA-2 strain background, respectively.

Example 2 Analysis of the A. niger PGOX-2 Derived Strains for the Amountof Glucose Oxidase Enzyme Product Produced

To be able to assess the effect of the GH gene disruptions, shake-flaskanalysis in FM medium of these transformants was analysed. At day 4 and6 after inoculation, medium samples were taken. The glucose oxidaselevels were analysed in the culture supernatant. For glucose oxidaseproduction, which is performed under control of the glucoamylasepromoter, surprisingly, a disruption of AgsE-An09g03070 resulted in anincreased production of glucose oxidase (FIG. 5), whereas the other twodisruptions of agdA and agdB showed no pronounced effect of glucoseoxidase production. Both the PGOX-2_AMY4-1 and PGOX-2_AMY4-2 strain, asidentified from glucose oxidase activities in the culture supernatant,had an increased activity on both sampling days (day 4 and 6). Thisincreased production upon AgsE disruption was confirmed by analyzing GOXexpression on plate (FIG. 6) for random transformants, which wereisolated as described in Example 1. These examples show that afilamentous fungal cell according to the invention has higher titers fora protein of interest in medium containing maltose as carbon source andis able to produce increased amounts of protein products in an AgsEdisrupted strain background.

Example 3 Analysis of the A. niger LIP-2 Derived Strains for the Amountof Lipolytic Enzyme L01 Product Produced

The AgsE gene disruption, which showed a positive effect on glucoseoxidase production in the PGOX-2 background, was further tested in theLIP-2 background. To be able to assess the effect of the AgsE genedisruption on lipase production, shake-flask analysis in FM medium ofthese transformants was analysed. At day 3-6 after inoculation, mediumsamples were taken. The lipase levels were analysed in the culturesupernatant; maximum productivity of the different strains was compared.For lipase production, which is performed under control of theglucoamylase promoter, surprisingly, disruption of AgsE-An09g03070resulted in an increased production of lipase (FIG. 7). Both theLIP-2_AMY4-1 and LIP-2_AMY4-2 strain, as identified from lipaseactivities in the culture supernatant, had an increased activity. Theseexamples show that a filamentous fungal cell according to the inventionhas higher titers for a protein of interest in medium containing maltoseas carbon source and is able to produce increased amounts of proteinproducts in an AgsE disrupted strain background.

Example 4 Analysis of the A. niger PLA-2 Derived Strains for the Amountof Phospholipase A2 Enzyme Product Produced

The AgsE gene disruption, which showed a positive effect both on glucoseoxidase production in the PGOX-2 background and on lipase production inthe in the LIP-2 background was tested further in the PLA-2 background.To be able to assess the effect of the AgsE gene disruption onphospholipase A2 production, 24-wells microtiterplate fermentation in FMmedium of these transformants was analysed. At 120 hours afterinoculation, medium samples were taken. The phospholipase A2 levels wereanalysed in the culture supernatant. For phospholipase A2 production,which is performed under control of the glucoamylase promoter,surprisingly, disruption of AgsE-An09g03070 resulted in an increasedproduction of phospholipase A2 (FIG. 8). These examples show that afilamentous fungal cell according to the invention has higher titers fora protein of interest in medium containing maltose as carbon source andis able to produce increased amounts of protein products in an AgsEdisrupted strain background.

The invention claimed is:
 1. A method for the production of a compoundof interest by microbial fermentation, wherein the compound of interestis selected from the group consisting of an enzyme and a metabolite, themethod comprising: a. providing a mutant filamentous fungal host cellcapable of expressing the compound of interest, wherein said mutantfilamentous fungal host cell has been modified in its genome to resultin a deficiency in the production of a polypeptide compared with aparent filamentous fungal host cell which has not been modified, andwherein both said mutant and said parent filamentous fungal host cell ismeasured under the same conditions, wherein the polypeptide is selectedfrom the group consisting of: i. a polypeptide comprising the amino acidsequence of SEQ ID NO: 3 or a polypeptide at least 90% identical theretoand having an enzymatic activity of the amino acid sequence of SEQ IDNO: 3; ii. a mature polypeptide comprised in the amino acid sequence ofSEQ ID NO: 3 or a polypeptide at least 90% identical thereto and havingan enzymatic activity of the mature polypeptide comprised in the aminoacid sequence of SEQ ID NO: 3; and iii. a polypeptide encoded by thepolynucleotide sequence of SEQ ID NO: 1 or 2 or encoded by apolynucleotide at least 90% identical to SEQ ID NO: 1 or 2, wherein saidpolypeptide encoded by a polynucleotide at least 90% identical to SEQ IDNO: 1 or 2 has an enzymatic activity of the polypeptide encoded by thepolynucleotide sequence of SEQ ID NO: 1 or 2; b. culturing said mutantfilamentous fungal host cell under conditions conducive to theexpression of the compound of interest, and c. optionally isolating thecompound of interest from the culture medium; wherein, in the mutantfilamentous fungal host cell, the modification in its genome is selectedfrom: A) a full or partial deletion of SEQ ID NO: 1 or a polynucleotideat least 90% identical to SEQ ID NO: 1; B) a full or partial replacementof SEQ ID NO: 1 or a polynucleotide at least 90% identical to SEQ ID NO:1 with a polynucleotide sequence which does not code for a polypeptideas defined in a) i. or a) ii. or with a polynucleotide sequence whichcodes for a partially or fully inactive form of a polypeptide as definedin a) i. or a) ii.; and C) a disruption of SEQ ID NO: 1 or apolynucleotide at least 90% identical to SEQ ID NO: 1 by the insertionof one or more nucleotides in the polynucleotide sequence and consequentpartial or full inactivation of a polypeptide as defined in a) i. or a)ii; wherein the polypeptide according to i. to iii. has an enzymaticactivity which is α-1,3-glucan synthase enzymatic activity; and whereinthe mutant filamentous fungal host cell further comprises at least onepolynucleotide coding for the compound of interest and/or at least onepolynucleotide coding for a polypeptide involved in the production ofthe compound of interest.
 2. The method according to claim 1, whereinthe yield of the compound of interest is increased compared to the yieldof a method for production of the compound of interest where the parentfilamentous fungal host cell which has not been modified is used, whenmeasured under the same conditions.
 3. The method according to claim 1,wherein, in the mutant filamentous fungal host cell, the maturepolypeptide comprised in the amino acid sequence of SEQ ID NO: 3 is themature polypeptide sequence of SEQ ID NO:
 4. 4. The method according toclaim 1, wherein, in the filamentous fungal host cell, the modificationin its genome is a full or partial deletion of SEQ ID NO: 1 or apolynucleotide at least 90% identical to SEQ ID NO:
 1. 5. The methodaccording to claim 1, wherein, in the mutant filamentous fungal hostcell, the modification in its genome is a disruption of SEQ ID NO: 1 ora polynucleotide at least 90% identical to SEQ ID NO: 1 by the insertionof one or more nucleotides in the polynucleotide sequence and consequentpartial or full inactivation of a polypeptide as defined in a.i. ora.ii.
 6. The method according to claim 1, wherein the mutant filamentousfungal host cell produces at least 5% less polypeptide as defined in a.i. to a. iii. compared with the parent filamentous fungal host cellwhich has not been modified, when measured under the same conditions. 7.The method according to claim 1, wherein the mutant filamentous fungalhost cell produces a polypeptide selected from a polypeptide at least90% identical to the amino acid sequence of SEQ ID NO: 3 and having anenzymatic activity of the amino acid sequence of SEQ ID NO: 3, apolypeptide at least 90% identical to the amino acid sequence of SEQ IDNO: 3 and having an enzymatic activity of the mature polypeptidecomprised in the amino acid sequence of SEQ ID NO: 3; a polypeptideencoded by the polynucleotide at least 90% identical to SEQ ID NO 1 or 2having an enzymatic activity of the polypeptide encoded by thepolynucleotide sequence of SEQ ID NO: 1 or 2, wherein said polypeptidehas at least 5% less enzymatic activity, compared with the parentfilamentous fungal host cell which has not been modified, when measuredunder the same conditions.
 8. The method according to claim 1, wherein,in the mutant filamentous fungal host cell, the modification in itsgenome is a full or partial replacement of SEQ ID NO: 1 or apolynucleotide at least 90% identical to SEQ ID NO: 1 with apolynucleotide sequence which does not code for a polypeptide as definedin a. i. or a. ii. or with a polynucleotide sequence which codes for apartially or fully inactive form of a polypeptide as defined in a. i. ora. ii.
 9. The method according to claim 1, wherein, in the mutantfilamentous fungal host cell, the modification which results in areduced or no production of a polypeptide as defined in i. to iii. isdue to a reduced or no production of the mRNA encoding said polypeptide.10. The method according to claim 1, wherein, in the mutant filamentousfungal host cell, the at least one polynucleotide coding for thecompound of interest and/or the at least one polynucleotide coding for acompound involved in the production of the compound of interest isoperably linked to a promoter.
 11. The method according to claim 10,wherein, in the mutant filamentous fungal host cell, the promoter is acarbohydrate inducible promoter.
 12. The method according to claim 1,wherein the mutant filamentous fungal host cell is selected fromAspergillus, Acremonium, Myceliophthora, Thielavia, Chrysosporium,Penicillium, Talaromyces, Rasamsonia, Fusarium and Trichoderma.
 13. Themethod according to claim 10, wherein the promoter is an induciblepromoter.
 14. The method according to claim 11, wherein the carbohydrateinducible promoter is selected from the group consisting of: a starchinducible promoter, a glucoamylase promoter, an acid stable amylasepromoter, an alpha-amylase promoter, and a TAKA amylase promoter. 15.The method according to claim 1, wherein the mutant filamentous fungalhost cell produces at least 99.9% less polypeptide as defined in a. i.to a. iii. compared with the parent filamentous fungal host cell whichhas not been modified, when measured under the same conditions.
 16. Themethod according to claim 1, wherein the mutant filamentous fungal hostcell is selected from Aspergillus niger, Aspergillus awamori,Aspergillus foetidus, Aspergillus sojae, Aspergillus fumigatus,Aspergillus oryzae, Acremonium alabamense, Myceliophthora thermophile,Thielavia terrestris, Chrysosporium lucknowense, Fusarium oxysporum,Rasamsonia emersonii, Talaromyces emersonii, Trichoderma reesei, andPenicillium chrysogenum.